Restriction Enzymes: A History

By Wil A.M. Loenen, Leiden University Medical Center
April 2019 · 346 pages, illustrated (38 color and 26 B&W)
ISBN 978-1-621821-05-2

<<  Chapter 6   —   Chapter 8  >>

Chapter 7

Chapter doi:10.1101/restrictionenzymes_7

Crystal Structures of Type II Restriction Enzymes and Discovery of the Common Core of the Catalytic Domain: ∼1993–2004


Chapter 6 used as starting material two reviews on the Type II and ATP-dependent (Type I and III) REases, published in the 1993 Nucleases book (Bickle 1993; Roberts and Halford 1993). This chapter, covering the next 10 years, is based on more than 25 reviews, reflecting the tremendous progress made during this period. Forty years after the first paper on EcoKI and EcoP1I (Bertani and Weigle 1953), reviews mentioned only about 20 Type I and Type III REases (Bickle and Krüger 1993; Roberts and Halford 1993), and because of lack of commercial interest, most research continued on EcoKI, EcoR124I, EcoP1I, and EcoP15I. Together, these two types comprised <2% of the enzymes identified in REBASE (Table 1); the newly named Type IV REases featured even less at 0.1% (and will be discussed in Chapter 8). This changed dramatically as, by the end of the century, improved sequencing and computer prediction programs showed R-M systems to be ubiquitous among Eubacteria and Archaea, with almost one-half of these genomes containing candidate Type II genes (Table 1; Roberts and Macelis 1991, 1993; Titheradge et al. 2001; Roberts et al. 2003b). To summarize these findings briefly, for which evidence had already become apparent (Chapter 6), there appeared to be extensive horizontal transfer of all R-M systems. Closely related systems were often present in unrelated organisms, and codon usage of R-M genes was often different from other host genes. In addition, the genes were found in different places on the genome in different strains of the same organism (Jeltsch and Pingoud 1996; Chinen et al. 2000; Nobusato et al. 2000a,b).


Table 1. Identified and candidate REases in REBASE in 2004

Enzymesa % Total Genomesb % Total

Type I


Type I


Type II


Type II


Type III


Type III


Type IV


Type IV


Courtesy of Rich Roberts; REBASE stats presented by Noreen Murray at the 5th NEB Meeting (Bristol 2004).

Total ∼3700 REases.

a Identified REase genes and characterized by biochemical assays.

b Candidate genes for REases in sequenced genomes.

By 2004, the number of REases had risen to ∼3700, making the family of REases a very large one indeed (Pingoud and Jeltsch 2001; Roberts et al. 2004). The dictum that Type II REases were simple dimeric enzymes, requiring Mg2+ (and no ATP or SAM) came under further scrutiny (Stasiak 1980a,b; Bennett and Halford 1989; Bujnicki 2000b; Murray 2000; Sapranauskas et al. 2000; Pingoud and Jeltsch 2001). Many new Type II enzymes recognized nonpalindromic DNA sites as monomers, tetramers, or higher-order complexes. Recognition sequences were often not unique, but could be discontinuous, degenerate, or asymmetric, whereas cleavage did not necessarily occur at the recognition site. The distinction into Type I, II, and III was still useful, but many REases clearly had intermediate properties, with no simple way to classify or predict DNA–protein interactions (Luscombe et al. 2001). What should one make of the many functional similarities, but also surprising diversity in DNA recognition and cleavage, and the positioning of metal cofactors (Aggarwal 1995; Wah et al. 1997; Viadiu and Aggarwal 1998; Pingoud and Jeltsch 2001)? Did this indicate highly subtle protein–DNA–cofactor interactions? The discovery of REases that had to interact with two copies of their recognition sequence before they could cleave DNA (Halford 2001) was exciting news. This process involved DNA looping, as reported for enzymes involved in replication, recombination, and transcription (Schleif 1992; Rippe et al. 1995). How could other REases that needed two sites in order to cleave be identified, and how did this cleavage occur? Would these REases perhaps be good tools to analyze interactions between distant DNA sites?

The rapid expansion of the REase family led to a book solely dedicated to “restriction enzymes” in 2004 (Pingoud 2004). The first chapter is a reprint of the Survey and Summary on the novel nomenclature of REases, MTases, Homing Endonucleases and their genes, published a year earlier (Roberts et al. 2003a). Edited by Alfred Pingoud, this book contains 16 additional chapters, including progress on EcoRI and EcoRV, as well as novel and often unexpected data on other Type II enzymes. Only one chapter is dedicated to the ATP-dependent Type I and III “molecular motors” (McClelland and Szczelkun 2004). A matter of strong debate was the issue of “selfishness,” especially that of the Type I and II systems (e.g., O'Neill et al. 1997; Kobayashi 1998). The nature of the catalytic core, the role of water and metal ions in mediating both the interaction of REases with their DNA recognition sites, and hydrolysis of the phosphodiester backbone (Cowan 2004; Sidorova and Rau 2004) were subject to intense study and fierce debate: How did the kind of metal cofactor (Mg2+, Mn2+, or Ca2+) influence the reactivity of the enzymes, and how many metal ions were needed? Another big question was whether it was possible to alter the specificity of EcoRI, EcoRV, BamHI, and other enzymes (Alves and Vennekohl 2004)? The elucidation of the structures of BamHI and BglII, which recognized sequences that differed only in the outer 2 base pairs, provided one explanation (Scheuring Vanamee et al. 2004). This disappointing immutability of REases contrasted with that of the mutability of transcription factors (Scheuring Vanamee et al. 2004). Expectations, however, were high with respect to the novel applications with chimeric REases, such as fusions with zinc fingers (Kandavelou et al. 2004).

Most data in the previous chapter concerned the mechanism of DNA specificity and cleavage of EcoRI and EcoRV, and reported the first crystal structures of EcoRI (Kim et al. 1990), EcoRV (Winkler et al. 1993), BamHI (Newman et al. 1994a,b), and PvuII with cognate DNA (Athanasiadis et al. 1994; Cheng et al. 1994). Twenty years after its initial characterization (Kuz'min et al. 1984; Schildkraut et al. 1984), by 2004, EcoRV was the most thoroughly studied REase (with the exception of EcoRI) through an elegant “pas de deux” of structural and mechanistic studies (Jen-Jacobson 1997; Winkler and Prota 2004). Toward the end of the century, there were reports on ∼1000 new Type II REases, and many biochemical and novel crystal studies (http://rebase.neb.com/rebase/rebase.html; Pingoud and Jeltsch 1997, 2001). By 2004, 16 Type II REases structures had been solved, plus those of four other nucleases and two resolvases (summarized in Table 1 of Horton et al. 2004a, p. 362). This included cocrystals of FokI, BglI, MunI, BglII, NgoMIV, BsoBI, and HincII (Wah et al. 1997; Newman et al. 1998; Deibert et al. 1999, 2000; Lukacs et al. 2000; van der Woerd et al. 2001; Horton et al. 2002). These new structures questioned the view held until the mid-1990s that the baffling lack of common features between most REases (in contrast to the MTases) suggested independent convergence, and not divergence from a common ancestor (Wilson 1991; Heitman 1993; Bujnicki 2004).

This chapter gives an overview of the period roughly from 1993 to 2004, during which the groups in Bristol, Pittsburgh, Edinburgh, and Basel continued their research into the biochemistry, structure, and relationships of the Type I, II, and III REases. NEB and Fermentas International, Inc. continued their search for novel enzymes, and investigations into the mechanisms of, and relationships between, these enzymes. In Germany, Alfred Pingoud moved from Hannover to Giessen, where he and Albert Jeltsch studied the structure, mechanism, and evolution of Type II REases (e.g., Pingoud and Jeltsch 1997, 2001; Jeltsch and Urbanke 2004). In Tokyo, Ichizo Kobayashi worked on his concept of R-M systems as “selfish” elements and minimal forms of life (Kobayashi 2004). Aneel Aggarwal in New York elucidated the surprisingly different structures of BamHI and BglII, mentioned previously (Scheuring Vanamee et al. 2004). In Berlin, Detlev Krüger and Monika Reuter investigated the reported, and puzzling, refractory EcoRII sites, even though these sites could be modified by M·EcoRII (Reuter et al. 2004). Virginijus (Virgis) Šikšnys in Vilnius started to unravel the structural and molecular mechanisms of sequence discrimination by REases recognizing closely related sequences (Šikšnys et al. 2004). In Warsaw, Janusz Bujnicki used the nine available crystal structures in combination with database searches to build evolutionary trees of the REase and nuclease superfamilies (Bujnicki 2004). Other aspects of the ATP-dependent Type I and III enzymes (Murray 2000, 2002; Dryden et al. 2001; Loenen 2003) and the Type II enzymes (Jeltsch and Pingoud 1996; Kovall and Matthews 1998, 1999; Bujnicki 2000a,b; Halford 2001; Mucke et al. 2003; Halford and Marko 2004; Kirsanova et al. 2004; Pingoud 2004; Pingoud et al. 2005) yielded valuable information and led to the new classification into 11 Type II subtypes in 2003, mentioned previously (Roberts et al. 2003a). Was the original idea of the function of REases too narrow, which had been based on the “arms race” of phages and conjugative plasmids to avoid restriction by counterattacks (Krüger and Bickle 1983; Bickle and Krüger 1993)? Would some of these REases perhaps have an additional role in recombination and transposition, rather than simply protect their host against foreign invaders (Arber 1979; Heitman 1993; McKane and Milkman 1995)?

For reasons of space, only a few examples can be discussed in this chapter and the reader is referred to the reviews for detailed information. Although the emphasis of this book is on REases, it should be mentioned that research into the MTases led to one of the most exciting discoveries of the 1990s—that is, base flipping. In the wake of the structure of M·HhaI (Cheng et al. 1993a,b), the M·HhaI–DNA complex revealed that the enzyme flipped the target base out of the DNA helix (Klimasauskas et al. 1994; Horton et al. 2004b). Base flipping would prove to be a more general property: Other MTases, endonucleases, and RNA enzymes “do it” (Winkler 1994; Mernagh et al. 1998; Roberts and Cheng 1998; Blumenthal and Cheng 2001; Cheng and Roberts 2001; Cheng and Blumenthal 2002; Su et al. 2005; Bochtler et al. 2006; Horton et al. 2006, 2014; Hashimoto et al. 2008). Other emerging interesting features of MTases such as the molecular evolution by circular permutations (e.g., Jeltsch 1999; Vilkaitis et al. 2002) would also be found in the HsdS subunits of Type I families (e.g., Loenen et al. 2014a).


Subtypes of Type II REases

In Chapter 6, several REases (e.g., EcoRII, NaeI, FokI, BcgI, and Sgr10I) were mentioned that were clearly not Type I or Type III, but also differed from the conventional Type II REases like EcoRI, EcoRV, and BamHI (Pingoud and Jeltsch 2001). With the discovery of many such new enzymes, Rich Roberts took the initiative to subdivide the Type II REases into 11 subtypes in 2003, being different from Type IIP (“P” for palindrome: EcoRI and EcoRV), Type IIS (FokI), and Type IIE (EcoRII, NaeI) (Table 2; Roberts et al. 2003a).


Table 2. Division of Type II REases in 11 subtypes in 2003

Subtypea Defining feature Examples Recognition sequence


Asymmetric recognition sequence


GGATG (9/13)




Cleaves both sides of target on both strands


(10/12) CGANNNNNNTGC(12/10)


Symmetric or asymmetric target. R and M functions in one polypeptide


CTGGAG (16/14)


(7/13) GAYNNNNNRTC (14/9)


(10/12) CGANNNNNNTGC (12/10)


Two targets; one cleaved, one an effector






Two targets; both cleaved coordinately






Symmetric or asymmetric target. Affected by AdoMet


GTGCAG (16/14)


CTGAAG (16/14)


Symmetric or asymmetric target. Similar to Type I gene structure


(10/12) CGANNNNNNTGC (12/10)




Subtype IIP or IIA. Require methylated target




Symmetric target and cleavage sites








Asymmetric target and cleavage sites


GGATG (9/13)


TCCRAC (20/18)


Symmetric or asymmetric target. R genes are heterodimers


CCTNAGC (−5/−2)b



Reprinted from Roberts et al. 2003a.

aNote that not all subtypes are mutually exclusive. E.g. BslI is of subtype P and T.

bThe abbreviation indicates double strand cleavage as shown below:



The initial definition of a Type II REase was that it cleaved at, or close to, the recognition site in an ATP-independent manner. The cleavage site could have a 5′ or 3′ sticky end (EcoRI, BglI) (Hedgpeth et al. 1972; Van Heuverswyn and Fiers 1980) or blunt/flush end (EcoRV) (Schildkraut et al. 1984). Type IIS (“S” for shifted) (e.g., FokI) was the first new subtype, named in 1991 (Szybalski et al. 1991). FokI had separate recognition and catalytic domains; the recognition domain had three smaller subdomains with helix-turn-helix (HTH) motifs, with the catalytic domain involved in potential dimerization (Wah et al. 1998). Initially thought to act as a monomer, FokI later proved to dimerize on the DNA (Bitinaite et al. 1998; Wah et al. 1998), now known to be not so unusual. EcoRII and NaeI were the first Type IIE subtypes, which interacted with two copies of their recognition sequence, one serving as allosteric effector (Krüger et al. 1988, 1995; Mucke et al. 2003). The Type IIF REases (e.g., SfiI and NgoMIV) also interacted with two copies of the recognition sequence but, in contrast to Type IIE, cleaved both sequences (Halford et al. 1999). Type IIT REases were heterodimeric proteins (e.g., Bpu10I and BslI) (Stankevicius et al. 1998; Hsieh et al. 2000). Type IIB were SAM-dependent heterodimeric REases, cleaving on both sides of an asymmetric recognition sequence (e.g., BcgI and BplI) (Kong and Smith 1997; Vitkute et al. 1997). In the case of “oddball” BcgI (Chapter 6), the catalytic centers for restriction and modification were located in the α subunit, with the DNA recognition domain in the β subunit (Kong 1998). Type IIG were single-chain SAM-dependent REases (e.g., Eco57I) (Janulaitis et al. 1992). Type IIM recognized methylated DNA (e.g., DpnI) (Lacks and Greenberg 1975). Other methylation-dependent REases were grouped as Type IV REases, the best-known enzyme being McrBC of E. coli (Raleigh and Wilson 1986; Stewart et al. 2000), which caused so much trouble in DNA cloning experiments (Raleigh et al. 1988). These enzymes were NTP-dependent (usually ATP, but GTP in the case of McrBC) for cleavage, like Type I and III REases, and cleavage occurred between sites (Stewart and Raleigh 1998; Panne et al. 1999). McrB was responsible for DNA recognition and GTP hydrolysis and McrC for catalysis (Pieper et al. 1999; Pieper and Pingoud 2002). Although a helpful subdivision, some enzymes fit into more than one category (for recent details, see http://rebase.neb.com/rebase/rebase.html; Loenen et al. 2014b) or in none of these properly (e.g., HaeIV) (Piekarowicz et al. 1999; Pingoud and Jeltsch 2001).

Two Types of Readout of Type II REases

The availability of more than 100 protein–DNA complex structures revealed two types of readout: direct readout of sequence via contacts with bases in the major (usually the most important) groove and in the minor groove, and indirect readout of sequence via interactions with the DNA backbone (Luscombe et al. 2001; Winkler and Prota 2004). In the presence of Mg2+, all Type II enzymes cleaved DNA with extremely high specificity (Roberts and Halford 1993). DNA sequences differing from the recognition site by just 1 bp were usually cleaved >106 times more slowly (Taylor and Halford 1989), far more than expected from the loss of a few H-bond interactions with a single base pair in a DNA–enzyme complex. Studies on EcoRI and other REases showed that total discrimination was always large (Lesser et al. 1990; Pingoud and Jeltsch 1997).

Type IIP: EcoRI and EcoRV

The wealth of crystal structures and biochemical studies with respect to EcoRI and EcoRV were extensively reviewed and are briefly summarized here (Pingoud and Jeltsch 2001; Grigorescu et al. 2004; Pingoud 2004; Winkler and Prota 2004).


John Rosenberg's group refined their data on the EcoRI structure (Kim et al. 1990), based on a high-resolution (1.85 Å) initial EcoRI–DNA recognition complex (Choi 1994), and a postreactive EcoRI–DNA complex at 2.7 Å resolution (using Mn2+) (reviewed in Grigorescu et al. 2004). The latter cocrystals were possible, as, fortuitously, in situ cleavage in the crystals could take place. More than a dozen different crystals with different oligonucleotides in the complex showed that EcoRI had a strong tendency to associate in sheets, and that the complex had twofold rotational symmetry axes perpendicular to the threefold axes (i.e., in the plane of the sheets) (Grable et al. 1984; Samudzi 1990; Wilkosz et al. 1995; Grigorescu et al. 2004). The protein became much more ordered after binding DNA, like EcoRV (see below). From earlier biochemical studies it was already known that the structure at the DNA–protein interface changed in response to even minor changes that were hard to predict (Lesser et al. 1990, 1993; Jen-Jacobson et al. 1991; Jen-Jacobson et al. 2000). Although EcoRI did bend the DNA like several other enzymes (Kim et al. 1994; Deibert et al. 1999; Lukacs et al. 2000; Pingoud and Jeltsch 2001), this was apparently not a general rule, as BamHI did not bend, kink, or unwind the DNA to any extent (Newman et al. 1995). None of the many EcoRI mutants altered the specificity of EcoRI for its recognition site, suggesting a general rule that mutations never led to a change of specificity, although catalytic activity might be severely impaired (Wolfes et al. 1986; Alves et al. 1989; Geiger et al. 1989; King et al. 1989; Needels et al. 1989; Wright et al. 1989; Hager et al. 1990; Heitman and Model 1990; Oelgeschläger et al. 1990; Jeltsch et al. 1993a; Flores et al. 1995; Grabowski et al. 1995; Muir et al. 1997; Windolph and Alves 1997; Fritz et al. 1998; Ivanenko et al. 1998; Kuster 1998; Rosati 1999; Grigorescu et al. 2004). Many amino acids in the main protein domain appeared essential for maintenance of the correct 3D structure of the dimer. In contrast, the promiscuous mutants (with reduced sequence specificity) (Chapter 6) localized to regions with low structural stability in the free enzyme (Heitman and Model 1990; Muir et al. 1997). Together with the available data on other REases, John Rosenberg concluded that no generalization could be made for the kind and extent of distortion Type II REases induced in their DNA substrate. In general, however, the DNA in the specific complex differed from ideal B-DNA, and distortions appeared to be part of the recognition process, as supported by the use of modified substrates or base analogs (e.g., Blattler et al. 1998).


Fritz Winkler's group refined the initial structure of EcoRV (Winkler et al. 1993) using many different crystals, revealing the initial EcoRV–DNA recognition complex, and the transition from nonspecific to specific complex with Mg2+, followed by DNA cleavage (Fig. 1; reviewed in Pingoud and Jeltsch 2001; Winkler and Prota 2004). The cleavage rate proved to be highly sensitive to interactions far from the active site. Major groove interactions were probed extensively using mutant enzymes, oligonucleotides, and modified bases (Fliess et al. 1988; Alves et al. 1989; Newman et al. 1990a,b; Thielking et al. 1991; Vermote and Halford 1992; Waters and Connolly 1994; Martin et al. 1999). Conflicting results were obtained with gel-shift, filter-binding, and steady-state fluorescence anisotropy techniques (Connolly et al. 2001). The gel shifts showed that EcoRV bound all sequences with equal affinity (Chapter 6), in contrast to EcoRI, suggesting a fundamentally different mechanism (Lesser et al. 1990; Thielking et al. 1990, 1992; Taylor et al. 1991; Vipond and Halford 1993, 1995; Winkler et al. 1993; Szczelkun and Connolly 1995). Conflict arose because the effect of Mg2+ on binding could not be analyzed directly because of rapid cleavage, a problem partly solved by using catalytically inactive mutants, Ca2+ as cofactor (which blocks cleavage), or using poor, or noncleavable, substrate analogs (Thielking et al. 1992; Winkler and Prota 2004). Was the interpretation of the EcoRV crystals correct? In the nonspecific complex, EcoRV bound not one, but two short DNA duplexes (of the self-complementary octamer CGAGCTCG) stacked end-to-end at the twofold axis, presumed to be representative for nonspecific binding (Winkler et al. 1993). This idea was challenged (Engler et al. 1997) and the issue reexamined (Erskine and Halford 1998; Reid et al. 2001). A decade later Winkler was still convinced that “the structure yields a very plausible explanation why no cleavage can occur in this binding mode” (Winkler and Prota 2004, p. 194).


FIGURE 1. Structure of EcoRV, free and in complex with nonspecific and specific DNA. The two subunits are shown in yellow and blue, respectively, and the DNA in red. On top of the complexes the DNA is shown at a right angle from the view below to illustrate the different degree of bending. (Reprinted from Winkler and Prota 2004, with permission from Springer Nature.)


The DNA-binding domain of EcoRV contained three segments, two of which interacted with the recognition site. This region contained a glutamine-rich “Q”-loop recognizing bases in the minor groove and a recognition “R”-loop making base-specific contacts in the major groove, presumably involved in cleaving both strands in one binding event (Thielking et al. 1991; Selent et al. 1992; Winkler et al. 1993; Kostrewa and Winkler 1995; Stahl et al. 1996, 1998b; Wenz et al. 1996; Thomas et al. 1999). The floor of the DNA-binding site appeared critical for coupling recognition and cleavage (Garcia et al. 1996). Some residues were involved in indirect readout, and others were relevant for conformational changes (Kostrewa and Winkler 1995; Thorogood et al. 1996; Wenz et al. 1996; Stahl et al. 1998a,b; Martin et al. 1999; Stanford et al. 1999). EcoRV had to open the DNA-binding site for the DNA to enter the cleft, similar to BamHI (Schulze et al. 1998; Viadiu and Aggarwal 2000).

This meant considerable conformational changes involving DNA bending and rotation of the DNA-binding domains, which wrapped around the DNA (Stover et al. 1993; Winkler et al. 1993; Kostrewa and Winkler 1995; Vipond and Halford 1995; Garcia et al. 1996; Horton and Perona 1998, 2000; Martin et al. 1999; Jones et al. 2001). EcoRV differed from EcoRI in the relative orientation of DNA and protein along the twofold symmetry axis, supporting the idea of “EcoRI-like” and “EcoRV-like” branches (Anderson 1993; Pingoud and Jeltsch 1997, 2001; Bujnicki 2000b; and below). What was the exact nature of this remarkable coupling of recognition and catalysis? The study of such changes warranted other tools and Winkler wondered whether single-molecule spectroscopy might lead to exciting new information on the relevance of the different structural states along the reaction path (Winkler and Prota 2004).

Type IIP: BamHI and BglII

The genes encoding BamHI from Bacillus amyloliquefaciens and BglII from B. subtilis subsp. globigii were finally cloned in the early 1990s (Brooks et al. 1991; Anton et al. 1997). Aneel Aggarwal's group set out to answer a key question: Did these two enzymes interact with DNA in the same way, as their recognition sequences differed by only the outer base pair (5′ G/GATCC and 5′ A/GATCT, respectively)? This analysis brought several surprises. The primary protein sequences proved to be unrelated, and, in contrast to all other known Type II REases, BamHI contained a critical glutamate as the third essential residue in the catalytic core (Selent et al. 1992; Dorner and Schildkraut 1994; Newman et al. 1994b; Grabowski et al. 1995; Lukacs et al. 2000). BglII proved unusual, too, because the catalytic residues were sequestered in a way not seen in any of the other REases.


Like EcoRI and EcoRV, BamHI derived its specificity from both binding and catalysis, and single base pair changes in the recognition site affected binding as much as a random sequence (Lesser et al. 1990; Thielking et al. 1990; Engler 1998; Engler et al. 2001). The preliminary structure (Chapter 6; Strzelecka et al. 1994) was followed by the structure of free enzyme, and with specific and nonspecific DNA (Newman et al. 1994b, 1995; Viadiu and Aggarwal 1998, 2000). Like EcoRI, BamHI could cleave the DNA in the crystals (Viadiu and Aggarwal 1998). The BamHI–DNA complex before cleavage was obtained using Ca2+, and after cleavage using Mn2+ (Fig. 2A; Scheuring Vanamee et al. 2004). As in most cases studied, the DNA was held in a tight-binding cleft (Aggarwal 1995; Pingoud and Jeltsch 1997, 2001). In the specific complex (Fig. 2Ac), DNA–protein interactions occurred both in the major and minor grooves (Newman et al. 1995; Scheuring Vanamee et al. 2004). Interestingly, the specific complex was asymmetrical, in contrast to the protein in the nonspecific complex. The carboxy-terminal arm of one subunit (called R) went into the DNA minor groove, whereas the arm from the other (L) subunit followed the DNA backbone. DNA cleavage occurred only in the R active site that contained two Mn2+ ions. In the nonspecific complex (Fig. 2Ab), the DNA protruded out of the cleft at the bottom of the BamHI dimer (Scheuring Vanamee et al. 2004). This complex would be highly competent for linear diffusion by sliding, as there were no base-specific contacts, and only a few water-mediated contacts to the phosphate backbone (Scheuring Vanamee et al. 2004). Therefore, this would prevent cleavage, like EcoRV in this situation (Winkler et al. 1993). However, in EcoRV the active site residues were displaced because of a change in DNA conformation, whereas in BamHI it was mainly because of a change in the protein conformation (Scheuring Vanamee et al. 2004).


FIGURE 2. The structures of BamHI and BglII. (A) Structure of (a) free, (b) nonspecific, and (c) DNA-bound forms of BamHI, respectively. Secondary structural elements, along with the amino terminus and carboxyl terminus, are labeled on the right monomer. Overall structure looking down the DNA axis. (B) Structure of (a) free and (b) DNA-bound forms of BglII: The enzyme is shown with its right subunit in the same orientation as the right subunit of the complex. Loops A and D and a part of loop E are disordered in the free enzyme and are drawn with dotted lines, corresponding to the conformation seen in the enzyme–DNA complex. The complex is viewed down the DNA axis. Secondary structural elements, along with the amino terminus and carboxyl terminus, are labeled on one monomer. Blue spheres mark the respective positions of Lys188 in the free and DNA-bound dimers. Each monomer swings by as much as ∼50°, like the blades of a pair of scissors, to open and close the binding cleft. The sheer magnitude of this motion is reflected by the dramatic increase in distances across the binding cleft. For example, the distance between symmetrically related Lys188 residues at the rim of the cleft increases from ∼17 Å in the complex to ∼61 Å in the free enzyme. (Reprinted from Scheuring Vanamee et al. 2004, with permission from Springer Nature.)


Overall, in the specific complex, the BamHI subunits clamped onto the DNA by an ∼10° rotation around the DNA axis moving in a tongs-like motion (Newman et al. 1995; Scheuring Vanamee et al. 2004). It was obvious why a DNA sequence with even a single wrong base pair would force the enzyme into a more open mode increasing the distance between the active site and scissile phosphate bonds. Thus, the enzyme could still bind to the nonspecific site (down by 102 to 103) but rarely cleave it (down by 107 to 1010) (Scheuring Vanamee et al. 2004). Based on the complexes with Ca2+ and Mn2+ ions, a two-metal mechanism was proposed for BamHI (Scheuring Vanamee et al. 2004), as discussed for E. coli DNA polymerase I (Beese and Steitz 1991). This proposal fit in with the finding that the metal binding sites in BamHI were superimposable on those of NgoMIV and with other calculations (Deibert et al. 2000; Fuxreiter and Osman 2001; Mordasini et al. 2003; Scheuring Vanamee et al. 2004). The structure of the nonspecific BamHI–DNA complex was the first to provide such a detailed picture of how an enzyme selected its specific site from the multitude of nonspecific sites (Scheuring Vanamee et al. 2004).

An issue that remained to be resolved was how BamHI would move along the DNA: Would a “corkscrew” motion of the enzyme along the DNA major groove follow initial nonspecific binding, or would the enzyme move along one face of the DNA (Sun et al. 2003)?


The structure of BglII turned out to be a big surprise (Scheuring Vanamee et al. 2004). In the specific complex, the DNA was completely encircled by the enzyme (Fig. 2Bb). The surface area buried upon DNA binding was much larger than in the BamHI complex. Another difference was that BglII distorted the DNA by bending ∼22° and by local unwinding and overwinding, similar to DNA complexes of EcoRI, EcoRV, and MunI (Kim et al. 1990; Winkler et al. 1993; Deibert et al. 1999). BglII opened up with a novel scissor-like motion to allow entry of the DNA, rather than binding the DNA in a tight cleft (Aggarwal 1995; Pingoud and Jeltsch 1997, 2001). This motion of the subunits was in a direction parallel rather than perpendicular to the DNA axis, as in the case of BamHI. To do this, the BglII monomers had to undergo a large motion to loosen their grip on the DNA. Interestingly, PvuII also completely encircled the DNA, but instead of a scissor-like motion, it opened with a tongs-like motion (Athanasiadis et al. 1994; Cheng et al. 1994; Scheuring Vanamee et al. 2004).

The large conformational change meant a change from a wedge-shaped bundle of α-helices in the free enzyme to a parallel four-helix bundle in the specific complex, which affected a so-called “lever” region. In the free enzyme, this lever was “down” hiding the catalytic site, whereas in the enzyme–DNA complex, this lever was “up,” exposing the catalytic residues for cleavage (Scheuring Vanamee et al. 2004). This was in contrast with BamHI, EcoRV, and PvuII, in which most of the active residues faced the solvent, but similar to free FokI, in which enzyme the cleavage domain was hidden by the recognition domain (Winkler et al. 1993; Athanasiadis et al. 1994; Newman et al. 1994a; Wah et al. 1997; Scheuring Vanamee et al. 2004).

Taken together, despite these differences in the way BamHI and BglII recognized the common base pairs, importantly, in both cases the whole protein contributed to the specificity. The structures explained why attempts to change the specificity of BamHI to that of BglII did not yield viable mutants (for discussion, see Scheuring Vanamee et al. 2004). Did this immutability reflect an evolutionary pressure not to look too much alike? With the benefit of hindsight, this would make sense: A simple change of specificity of the REase through a few point mutations would mean that the cognate MTase could no longer protect the host DNA against restriction. This would put pressure on the REases to develop an intimate relationship with the recognition site that could not easily be changed. By 2004, only EcoRV and BamHI had been analyzed in a specific and nonspecific complex, showing obvious common features (Figs. 1 and 2A). Although tempted, Aneel Aggarwal cautioned that it was still too early to make general statements regarding structural changes accompanying the transition from nonspecific to specific binding, based on only these two enzymes (Scheuring Vanamee et al. 2004).

Type IIE REases: EcoRII and NaeI

EcoRII was one of the first Type II R-M systems to be discovered, and cleaved 5′ C/CWGG (Arber and Morse 1965; Bannister and Glover 1968, 1970; Takano et al. 1968; Yoshimori et al. 1972; Bigger et al. 1973; Boyer et al. 1973). But surprisingly, phage T3 DNA was resistant to EcoRII cleavage although not modified by Dcm and cut by isoschizomer BstNI (5′ CC/WGG) (Krüger et al. 1985,1988). What was going on?

The answer to this question came from an unusual experiment: The addition of pBR322 DNA (a plasmid with six EcoRII sites) to the refractory T3-EcoRII digestion mixture allowed restriction of T3 DNA (Fig. 3, lanes 4 and 6). Complete cleavage required a molar ratio of 2:1 (Pein et al. 1989). Synthetic 14-bp oligonucleotide duplexes (but not ssDNA) with a single EcoRII site could also activate cleavage of T3 DNA by EcoRII, at a molar ratio of 140:1 (Pein et al. 1989). The 14-bp duplexes were cleaved themselves as well, indicating that EcoRII could simultaneously bring together two molecules in an enzyme–DNA complex.


FIGURE 3. Activation in trans by pBR322 DNA for cleavage of refractory sites in T3 DNA by the first Type IIE REase, EcoRII, an enzyme that needs two sites for cleavage (Krüger et al. 1988). From left to right: 1 kb ladder (lane 1), T3 DNA (∼40 kb, lane 2), T3 DNA treated with BstNI (lane 3) or EcoRII (lane 4), pBR322 DNA treated with EcoRII (lane 5), and a mixture of T3 DNA and pBR322 DNA treated with EcoRII (lane 6). T3 bands are labeled A, B, C, D; pBR322 band in base pairs. The fragments were separated on 0.7% agarose gel (adapted from Krüger et al. 1988; for technical reasons Dcm DNA was used, which will not be explained here). (Reprinted from Krüger et al. 1988.)


Similar resistance was found for phage T7 DNA, whereas that of phage f1 RF dsDNA was incomplete, although modification of ssDNA was possible (Arber 1966; Hattman 1973; Vovis et al. 1975; Krüger et al. 1988). EcoRII was the first example of a REase that could bind two copies of its DNA recognition sequence in trans. The next question was, if EcoRII interacted simultaneously with two DNA sites, did the activating DNA molecules necessarily have to be cleavable themselves? To address this issue, pBR322 DNA was cut with EcoRII, and after phenol extraction and ethanol precipitation, this DNA was incubated with T3 DNA and EcoRII. The results were clear: The EcoRII-derived pBR322 cleavage products stimulated T3 cleavage, but those derived from isoschizomer BstNI (or MvaI) did not. This proved that (1) cleavage of the activating pBR322 EcoRII sites themselves was not necessary, and, interestingly, (2) the nature of the sticky ends mattered (Pein et al. 1991).

A second enzyme requiring activation in trans was NaeI from Nocardia aerocolonigenes (Conrad and Topal 1989). Both EcoRII and NaeI were dimers in solution and became the prototypes of the Type IIE REases, whose subtype also includes an enzyme of recent interest, SgrAI (Kosykh et al. 1982; Krüger et al. 1988; Conrad and Topal 1989; Vinogradova et al. 1990; Baxter and Topal 1993; Bitinaite and Schildkraut 2002; Roberts et al. 2003a; Dryden 2013). In both cases, separate catalytic and DNA recognition domains bound the two copies of the recognition site simultaneously (Krüger et al. 1988; Conrad and Topal 1989; Colandene and Topal 1998). In the case of EcoRII, the amino-terminal domain controlled the need for two sites to allow catalytic activity of the carboxy-terminal domain: The latter domain could be separately purified as a dimer, and then could cleave DNA with a single EcoRII site (Mucke et al. 2002; Zhou et al. 2004). EcoRII independently cleaved both strands in a single binding event (Yolov et al. 1985; Petrauskene et al. 1998). Activator duplexes of 14 bp were poorly cleaved at low concentrations, but were good activators and substrates at high concentrations, indicating positive cooperativity (Gabbara and Bhagwat 1992). Oligonucleotide duplexes with modified bases (phosphorothioate at the cleavage position) could still activate without being cleaved themselves (Pein et al. 1991; Conrad and Topal 1992; Senesac and Allen 1995). In the case of NaeI, this finding led to the commercial use of NaeI as “Turbo NaeI” (Senesac and Allen 1995; Reuter et al. 2004). There appeared to be three types of sites: resistant, slow, and cleavable substrate sites; the former two were stimulated by either activator DNA or spermidine (Oller et al. 1991). Also, EcoRII cleaved duplexes of increasing length (14, 30, and 71 bp) with decreasing efficiency, in contrast to isoschizomer MvaI (Cech et al. 1988; Pein et al. 1991).

DNA loops in cis could be seen by EM (Topal et al. 1991; Mucke et al. 2000), but they also occurred in trans (Krüger et al. 1988; Conrad and Topal 1989; Pein et al. 1989, 1991; Gabbara and Bhagwat 1992; Piatrauskene et al. 1996). Interaction with two recognition sites could be achieved either by one dimer alone or by binding of one dimer per site and subsequent formation of an active tetrameric protein–DNA complex. Although able to act in trans, EcoRII and NaeI both preferred interactions in cis, forming loops with DNA molecules with two or more sites <1 kb away from each other (Krüger et al. 1988; Pein et al. 1991; Schleif 1992). This tied in with other reports that interactions between DNA sites in cis were usually favored over those in trans (Schleif 1992; Rippe et al. 1995).

NaeI had two nonequivalent DNA-binding sites: The recognition domain would bind one recognition site—the activator site; this activated the catalytic domain, enabling cleavage of the other site; the second DNA recognition site was required for efficient cleavage (Oller et al. 1991; Gabbara and Bhagwat 1992; Yang and Topal 1992; Kupper et al. 1995; Colandene and Topal 1998; Reuter et al. 1999; Huai et al. 2000, 2001; Mucke et al. 2002). This was shown using different plasmids with one or two sites (Embleton et al. 2001; Mucke et al. 2003). Catenanes (Fig. 4) could be used to test whether an enzyme used sliding along the DNA (1D tracking) to find its recognition site or used 3D looping (Adzuma and Mizuuchi 1989). Such interlinked rings with a copy of the target site in each of the two rings could be generated using resolvase on a plasmid with two res sites interspersed with two targets for the enzyme under study. Although a looping enzyme can move from its site to the site on the other ring, a tracking enzyme will be unable to transfer to the other ring. Type IIE enzymes cleaved only one ring of the catenanes (Embleton et al. 2001), in contrast to the Type IIF REases (Szczelkun and Halford 1996; see next subsection).


FIGURE 4. Use of catenanes to study tracking and looping by REases. A plasmid contains two target sites for resolvase in direct repeat, the res sites (black triangles), and two recognition sites (hatch marks) for a protein that can bind concurrently to two sites (e.g., a Type IIE REase). The binding of this protein (shown as four spheres) to both sites sequesters the res sites into separate loops. In the absence of this binding protein, resolvase acts on the plasmid to yield a DNA catenane. However, resolvase is blocked if the res sites are sequestered in separate loops. (Reprinted from Welsh et al. 2004, with permission from Springer Nature; originally adapted from Milsom et al. 2001, with permission from Elsevier.)


The crystal structure of NaeI revealed a structural motif for the DNA-binding site occurring in the catabolite activator protein that was not present in EcoRII (Huai et al. 2000, 2001; Zhou et al. 2004). In the absence of DNA, only the catalytic domain of NaeI contributed to dimerization, whereas, in the presence of activator DNA, the DNA-binding domain also contributed, resulting in a more compact dimer (Huai et al. 2000, 2001). Apparently, NaeI changed conformation after binding activator DNA, which promoted binding of, and cleavage by, the catalytic domain of the DNA, the active complex being a protein dimer bound to two DNA recognition sites (Petrauskene et al. 1994; Reuter et al. 1998; Huai et al. 2000, 2001; Mucke et al. 2002).

The differences between the DNA-binding domains of EcoRII and NaeI suggested that different Type IIE enzymes had independently (i.e., “convergently”) found a similar solution to the same problem of binding two identical DNA sites.

Type IIF REases: SfiI, Cfr10I, and NgoMIV

Like Type IIE REases, SfiI, Cfr10I, and NgoMIV bound simultaneously to two DNA sites, but were classed as subtype IIF as they acted at both sites at the same time, and converted catenanes with two sites directly to two linear products, unlike Type IIE (Wentzell et al. 1995; Szczelkun and Halford 1996; Šiksnys et al. 1999; Embleton et al. 2001; Roberts et al. 2003a). To align two sites for cleavage, these enzymes may require a certain length of the intervening DNA. Would it have consequences for the activity of the protein, if one altered the length of the nonspecific spacer by 5–6 bp, or 10–11 bp? In the former case, such a change would rotate one recognition surface relative to the other by about 180°, requiring (under- or over-)twisting the intervening DNA to align the sites, an energetically costly event. In contrast, an alteration of 10–11 bp would simply add an additional helical turn. If so, would variations in the length of the spacer show a cyclical response characteristic of DNA looping, with a periodicity expected for the helical repeat (Schleif 1992)? Indeed, SfiI (GGCC[N5]GGCC) did show such a cyclical response when tested against plasmids with two SfiI sites separated by various lengths of DNA of <300 bp (Wentzell and Halford 1998; Welsh et al. 2004).

NgoMIV, Bse634I, and Cfr10I

Around 1995, the group of Virgis Šikšnys started the analysis of NgoMIV, and several other REases that recognized 5′ CCGG in different contexts, but belonged to different subtypes. SsoII and StyD4I (IIP), EcoRII (IIE), and NgoMIV (IIF), appeared to possess a similar DNA-binding motif and catalytic center: Were these subfamilies perhaps evolutionary related, and did they share a common ancestor, probably a homodimer (Pingoud et al. 2002; Tamulaitis et al. 2002)? The first three crystal structures analyzed were NgoMIV from Neisseria gonorrhoeae (recognition site 5′ G/CCGGC) (Stein et al. 1992) and two isoschizomers that shared ∼30% identity: Cfr10I from C. freundii and Bse634I from Bacillus stearothermophiles (recognition site 5′ Pu/CCGGPy) (Janulaitis et al. 1983; Repin et al. 1995; Grazulis et al. 2002). The structures of Cfr10I and Bse634I without DNA (Bozic et al. 1996; Grazulis et al. 2002) and that of NgoMIV with DNA (Deibert et al. 2000) proved that these enzymes acted as tetramers (although Cfr10I was initially thought to be a dimer as the dimer–dimer interface was considered to be due to crystal packing). Two monomers formed a primary dimer similar to that of Type IIP enzymes such as EcoRI (Fig. 5A; Rosenberg 1991). This similarity supported the notion of a common core and active site but also the idea that perhaps the cleavage pattern rather than the recognition sequence played a key role in the structure of the dimer (Anderson 1993; Aggarwal 1995).


FIGURE 5. (A) Primary dimers of the tetrameric REases Cfr10I, Bse634I, and NgoMIV and comparison with EcoRI. Individual subunits are shown in gray and black. (B) Tetramers of Cfr10I, Bse634I, and NgoMIV. Two back-to-back primary dimers are shown in gray and black. The monomers are labeled A, B, C, D, respectively. DNA molecules bound to NgoMIV are shown in the stick presentation. (Reprinted from Šikšnys et al. 2004, with permission from Springer Nature.)


The specific complex of NgoMIV with two 10-bp oligonucleotide duplexes showed two primary dimers back-to-back with the DNA on the opposite sides of a tetramer, with the major groove contacts between the dimer and the recognition site. The DNA recognition and dimerization interfaces in NgoMIV were intertwined, and the tetramer was fixed by contacts between both subunits and primary dimers (Šikšnys et al. 2004). The most extensive contacts were located in the “tetramerization” loop. A single mutation in this region, and parallel experiments in solution, showed that being a tetramer was important for restriction (Bilcock and Halford 1999; Šikšnys et al. 1999; Deibert et al. 2000; Milsom et al. 2001; Pingoud and Jeltsch 2001; Grazulis et al. 2002; Šikšnys et al. 2004). Using the above catenane assays, simultaneous cleavage of all four bonds by NgoMIV, Bse634I, and Cfr10I was confirmed (Bilcock et al. 1999; Bath et al. 2002; Šikšnys et al. 2004). Slow cleavage of single-site plasmids could be speeded up by oligonucleotide duplexes with (but not without) the recognition sequence, similar to transactivation of SfiI (Nobbs and Halford 1995).

Modeling of the structures of free Bse634I and Cfr10I on the NgoMIV–DNA complex indicated similar recognition of the central CCGG, but not the outer base pair (Grazulis et al. 2002; Šikšnys et al. 2004). This resembled the pattern shown for EcoRI (G/AATTC) and MunI (C/AATTG) (Kim et al. 1990; Šikšnys et al. 1994; Jen-Jacobson et al. 1996; Deibert et al. 1999; Lukacs and Aggarwal 2001), but not BamHI and BglII (see above, and Lukacs et al. 2000; Lukacs and Aggarwal 2001; Scheuring Vanamee et al. 2004). Interestingly, in the crystals, the amino acids contacting the two Mg2+ ions were in the same relative location, although they were derived from different regions of the polypeptide (Skirgaila et al. 1998; Deibert et al. 2000; Grazulis et al. 2002). This suggested plasticity of the active sites as long as the structure was conserved, rather than the primary sequence, as also reported for other enzymes (Todd et al. 2002). This was supported by a residue swapping experiment of Cfr10I, which created a reengineered metal binding site with significant catalytic activity (Skirgaila et al. 1998).

Computer and sequence analysis suggested that NgoMIV, Cfr10I, and Bse634I shared a common ancestor with SsoII and PspGI, but in these enzymes the orientation of the monomers in the dimer had changed, and their pentanucleotide 5′ sticky ends were the result of an increased distance between the two catalytic sites compared to NgoMIV (Pingoud et al. 2002; Bujnicki 2004). Was this perhaps a common evolutionary mechanism for the generation of new specificities?

NgoMIV, Cfr10I, and Bse634I were the first Type IIF enzymes to be crystallized, ahead of the structure of the archetype Type IIF enzyme, SfiI (Viadiu et al. 2003; Vanamee et al. 2005). Sfi I serves as a model enzyme to study how two DNA molecules can be sequestered in a synaptic complex, an event that is used for many reactions in the cell. Analysis of how this enzyme recognizes and cleaves its target DNA would provide insight into the sequential binding events that result in such a complex. The sequence of Sfi appeared to be totally unrelated to other proteins, but its mode of DNA recognition is similar to that of the dimeric Type IIP BglI enzyme, even though SfiI is a tetramer (Vanamee et al. 2005). Bioinformatic analysis supported the notion that SfiI was more closely related to BglI than to any other REase, including other Type IIF REases with known structures, such as NgoMIV·NgoMIV and BglI were judged to belong to two different, very remotely related branches of the PD· (D/E)XK superfamily: the α class (EcoRI-like) and the β class (EcoRV-like), respectively. This analysis provided “evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD…(D/E)XK superfamily of REases.” The model of SfiI would be useful for further experimental analyses.

The Common Core of the Majority of the Type II REases

The studies in Vilnius provided the first formal evidence for the conserved sequence motif in the active site of REases, now known as the PD…(D/E)XK site (Fig. 6).


FIGURE 6. Structural localization of the active site residues of EcoRI, NgoMIV, and Cfr10I. Conserved structural elements are shown in stick representation and labeled. Mn2+ ion present in the active site of EcoRI (PDB entry 1 qps) is shown as a gray sphere. Two Mg2+ ions present in NgoMIV are shown as gray spheres. Active site motifs corresponding to the first metal ion binding site are shown below each figure. Arrows indicate Cfr10I active site residues subjected to swapping (see text; Skirgaila et al. 1998). (Reprinted from Šikšnys et al. 2004, with permission from Springer Nature.)


This motif appeared to be common to 11 other REases belonging to Type IIP, IIE, and IIF (Fig. 7; Kovall and Matthews 1999; Pingoud et al. 2002; Šikšnys et al. 2004). This core had already been noted, when the structures of EcoRI and EcoRV were compared (Venclovas et al. 1994). A five-stranded mixed β-sheet was flanked by α-helices, also present in four other endonucleases: lambda exonuclease, MutH, Vsr endonuclease, and TnsA (Ban and Yang 1998; Kovall and Matthews 1999; Tsutakawa et al. 1999a; Hickman et al. 2000). Of the four β-strands, three β-strands were absolutely conserved. Within this common core, two β-strands would be directly involved in catalysis, and the others would be important for the structure itself. Was there a common nuclease ancestor, which had been subject to divergent evolution, as long as this structure remained intact (Huai et al. 2000)?


FIGURE 7. Comparison of the REase folds in some members of the REase superfamily. The conserved central β-sheet fold is highlighted in dark gray. The catalytic residues are in ball-and-stick and colored black. Single molecules of BamHI, EcoRI, EcoRV, PvuII, and Cfr10I and only the amino-terminal catalytic domains of FokI and TnsA are shown. (Reprinted from Horton et al. 2004a, with permission from Springer Nature.)


Mutants in the PD ··· (D/E)XK motif of EcoRV confirmed the essential role of these residues (Thielking et al. 1991; Winkler 1992). Outside this PD…(D/E)XK fold, different REases would have acquired different additional elements (e.g., Type IIF REases a tetramerization region and Type IIE REases a domain for binding two sites). This idea was supported by mutations in the respective regions (Reuter et al. 1999; Šikšnys et al. 1999, 2004; Deibert et al. 2000; Mucke et al. 2002; Bujnicki 2004; Zaremba et al. 2005, 2006, 2012). Did the idea of evolutionary relationships between different subtypes suggest an abrupt or continuous transition? Was this independent of different higher-order tertiary and quaternary structures and despite different substrate requirements for DNA cleavage (Pingoud et al. 2002; Tamulaitis et al. 2002)? Similarity at the tertiary structure was strongest between REases with a similar cleavage pattern—for example, BamHI and EcoRI (four-base 5′ overhang, DNA binding from the major groove side) or EcoRV and PvuII (blunt end, DNA binding from the minor groove side) (Anderson 1993; Aggarwal 1995). Again, this indicated that the nature of the DNA cleavage site was important, rather than the recognition sequence (Anderson 1993; Aggarwal 1995). An exception was BglI: It had a fold similar to EcoRV and PvuII, but it cleaved DNA to leave a 3′ overhang (Newman et al. 1998). This difference could be “explained away” by relatively minor modifications of the protein surface (Newman et al. 1998). It was concluded that two families of enzymes could be distinguished that were structurally very similar: EcoRI-like enzymes and EcoRV-like enzymes. The EcoRI-like REases usually recognized specific bases in the DNA mainly via residues from an α-helix, whereas the EcoRV-like REases usually recognized specific bases in the DNA mainly via residues from an additional β-sheet (Fig. 8; Aggarwal 1995; Bujnicki 2000b, 2001b; Huai et al. 2000; Pingoud and Jeltsch 2001). Despite this, the two families of enzymes were structurally very similar (see Table 1 in Horton et al. 2004a for details, pp. 362–363).


FIGURE 8. Diagrams showing the major structural differences between the α (EcoRI-like) and β (EcoRV-like) subclasses of the PD…(D/E)XK enzymes (Huai et al. 2000; Bujnicki 2001b). Common secondary structures are shown in black. Key elements involved in DNA recognition are shown in gray (in α class it is a universally conserved α-helix B; in β class it is an additional small β-sheet). Other elements specific for α and β subclasses (including the topologically fifth β-strand) are shown in white. The alternative site in α-helix B, to which the D/E carboxylate migrated in some of the enzymes from the α class, is indicated as “alt.” (Modified with permission of Bentham Science Publishers, Ltd., from Bujnicki 2003.)


The fact that Type II subtypes shared the PD…(D/E)XK motif suggested a basically similar reaction mechanism, but there was at least one exception: BfiI was Type IIS like FokI, but used a “zero-metal” mechanism (Sapranauskas et al. 2000; Zaremba et al. 2004). Surprisingly, BfiI belonged to the phospholipase D (PLD) superfamily and resembled NucA from S. typhimurium, whose crystal structure was known (Stuckey and Dixon 1999). As NucA was a homodimer with one catalytic center formed by the two subunits, perhaps BfiI would also form a tetramer for double-strand cleavage (Pingoud and Jeltsch 2001). More exceptions like BfiI were likely to follow based on other sequence comparisons, which suggested that some Type II REases belonged to the HNH and GIY-YIG families of endonucleases (Aravind et al. 2000; Bujnicki et al. 2001).

Evolutionary Relationships between REases

Janusz Bujnicki used the method of Johnson and coworkers (Johnson et al. 1990) and the atomic coordinates of the nine available REase structures to propose an evolutionary tree (Fig. 9; Bujnicki 2000b).


FIGURE 9. Phylogenetic tree of the PD…(D/E)XK superfamily, based on the “structural tree” (Bujnicki 2004, p. 76), and expanded to include additional members, identified by sequence analyses and protein-fold recognition (Šikšnys et al. 1995; Aravind et al. 2000; Bujnicki 2001a; Bujnicki and Rychlewski 2001a,b; Friedhoff et al. 2001; Pingoud et al. 2002; Rigden et al. 2002). REases are shown in black frames. PD…(D/E)XK domains identified by bioinformatics and not by crystallography are shown in white on gray background. Subtype IIE enzymes from three different lineages are indicated by circles. Isoschizomers MboI and Sau3A that originated from two different lineages are indicated by a label with their recognition site GATC. Parts of the tree that could not be confidently resolved based on either sequence or structural analysis are shown in broken lines. (Reprinted from Bujnicki 2004, with permission from Springer Nature.)


A comparison of crystal structures of REases with other proteins suggested that they were related to other DNA processing proteins, including DNA recombinases and transcription factors, which formed loops in the DNA depending on the length of the DNA between recognition sites, as first reported in 1984 (Dunn et al. 1984; Topal et al. 1991; Ban and Yang 1998; Wentzell and Halford 1998; Kovall and Matthews 1999; Tsutakawa et al. 1999b; Hickman et al. 2000). EcoRII and NaeI shared motifs with some site-specific recombinases, and, in line with this, a single mutation in EcoRII and NaeI turned these enzymes into topoisomerases (Topal and Conrad 1993; Jo and Topal 1995; Nunes-Duby et al. 1998; Carrick and Topal 2003). Extensions or insertions in regions outside the common PD…(D/E)XK fold of SsoII, PspGI, NgoMIV, and EcoRII appeared to determine the features characteristic for the different subtypes mentioned previously (IIP, IIF, or IIE) (Reuter et al. 1999; Deibert et al. 2000; Bujnicki 2004). Sau3AI was a highly unusual enzyme with similarity to MutH: It bound two recognition sites and formed DNA loops, like Type IIE and IIF, but used two copies of a duplicated PD…(D/E)XK domain, of which only the amino-terminal copy retained the conserved catalytic residues (Bujnicki 2001a; Friedhoff et al. 2001). The Type IIS REase FokI interacted with two sites via a domain resembling that of Tn7 transposase (TnsA) (Hickman et al. 2000), whereas DNA excision by SfiI was reminiscent of recombinases that simultaneously cleaved four DNA strands (Wentzell and Halford 1998). This structural and functional similarity with recombinases and transposases led to speculations that REases might promote genetic rearrangements and enhance genome diversity (Carlson and Kosturko 1998; Hickman et al. 2000; Mucke et al. 2002). REases would thus benefit the population as a whole, rather than individual organisms or the R-M systems themselves, as Tom Bickle had already pondered (Chapter 6; Bickle 1993; Arber 2000). In the absence of in vivo or in vitro evidence (Petrauskene et al. 1998; Šikšnys et al. 1999; Deibert et al. 2000), by 2004, the tentative general conclusion seemed to be that R-M systems could integrate DNA fragments into the genome of the host but could also shuffle protein domains around as a neat way to create new functions. Interestingly, this was apparently not limited to bacteria and their plasmids, viruses, and transposons, as genes could also pass from vertebrates into bacteria (Ponting and Russell 2002).

The Role of Water in Specific and Nonspecific Recognition by Type II REases

Nina Sidorova and Donald Rau analyzed the role of H2O in specific and nonspecific recognition by EcoRI (Sidorova and Rau 1996, 2004; Pingoud 2004), important in light of the effect of hydrostatic and osmotic pressure on the activity of REases (e.g., that on Type IV Mrr; Ghosh et al. 2014). As these studies require further analysis, the reader is referred to the original review for further information on this topic (Sidorova and Rau 2004).

Role of Mg2+ and Other Metal Cofactors of Type II REases

Most REase cleavage studies were consistent with a direct attack by H2O and absence of an intermediate species, as shown by inversion of the stereochemistry at phosphorus (Connolly et al. 1984; Grasby and Connolly 1992; Mizuuchi et al. 1999), in contrast to BfiI that goes via a covalent complex and thus retention of stereo configuration as mentioned previously. Different groups proposed models to explain the metal dependence in this process (Jeltsch et al. 1992, 1995b; Baldwin et al. 1995; Vipond et al. 1995; Vipond and Halford 1995; Horton et al. 1998a). Usually, REase activity first increased with increasing [Mg2+], but then dropped, possibly as a result of substrate inhibition, most likely because of general ionic strength effects or competition between the metal ions (Demple et al. 1986; Black and Cowan 1994; Friedhoff et al. 1996). Interestingly, polyamines diminished this inhibition by displacing Mg2+ ions (Friedhoff et al. 1996). But what made Mg2+ so special? According to Cowan in Columbus, Ohio, Mg2+ was “a good choice due to its high abundance, and a favorable combination of physical and chemical properties” (Cowan 2004). These properties included a tendency to bind H2O molecules rather than bulkier ligands (Cowan 2004). Effectively, Mg2+ usually interacted with two to three oxygens on REase side chains, unlike other metals such as Mn2+ (Cowan 1998, 2004). In strong contrast, Ca2+ blocked cleavage, a useful property in the crystal studies discussed previously (Jose et al. 1999; Conlan and Dupureur 2002a; Cowan 2004).

Everybody agreed that the elucidation of the mechanism of DNA cleavage critically depended on the number of Mg2+ ions directly involved in catalysis. Alfred Pingoud and coworkers proposed a one-metal mechanism for EcoRV catalysis but could not exclude a two-metal mechanism (Jeltsch et al. 1992, 1993b). A heated debate continued for decades: How many metal ions were needed for catalysis: one, two, or three (Jeltsch et al. 1992; Vipond et al. 1995; Cowan 1998; Lukacs et al. 2000; Chevalier et al. 2001)? The issue remains unresolved until today (The Seventh NEB Meeting 2015). This was not helped by the “perplexing” observation that Mg2+, Ca2+, and Mn2+ did not necessarily bind to the same amino acid side chains within the catalytic core (Pingoud and Jeltsch 2001). For EcoRV, at least three different mechanisms were proposed, based on combined data from different crystal structures (Kostrewa and Winkler 1995; Pingoud and Jeltsch 1997, 2001; Horton et al. 1998b; Kovall and Matthews 1999; Cowan 2004; Horton et al. 2004a). How misleading were these structures? Was the three-metal catalytic model the result of movement of the metal(s) during the transition from nonspecific to specific binding, and positioning the catalytic site of the enzyme near the bond to cleave (Kostrewa and Winkler 1995; Cowan 2004)? Perhaps the crystal structures of EcoRV were “snapshots” along the reaction pathway (Horton et al. 2004a)!

In the case of PvuII, Dupureur and coworkers showed a conformational change after metal ion binding and decided on a two-metal mechanism (Conlan et al. 1999; Dupureur and Hallman 1999; Dupureur and Conlan 2000; Dominguez et al. 2001; Dupureur and Dominguez 2001; Conlan and Dupureur 2002a,b). PvuII–DNA binding was only promoted by metal ions for specific recognition (Conlan and Dupureur 2002a,b), suggesting that “the placement of the metal cofactor is optimal to promote specific contacts with the cognate sequence, either through direct binding interactions or an indirect influence on enzyme structure” (Cowan 2004). Did the enzyme need two metal ions located close together (<∼4 Å) on two sides of the substrate, as proposed for DNA polymerase I (Beese and Steitz 1991; Cowan 1998)? And did the overall data support the notion that one metal ion would promote cleavage, whereas the other one (or both) would serve a structural role and/or influence substrate binding (Cowan 2004)? In this process each metal ion would influence the binding of the other (Cowan 2004). Evidently more kinetic studies were needed under solution turnover conditions with Mg2+ as cofactor; not an easy task, but vital to solve this problem and put an end to the metal debate (Cowan 2004).

Engineering and Applications of Chimeric Type II REases

A wide variety of mutations were introduced in various REases, ranging from mutant enzymes with enhanced cleavage or relaxed specificity, recognition of altered or modified sequences, and lengthening of the recognition site to changed subunit composition and single-chain nucleases (summarized in Table 1, pp. 394–395 of Alves and Vennekohl 2004). Unfortunately, REases appeared to require many changes in order to generate REases with new specificities (Jeltsch et al. 1995a; Anton et al. 1997; Bujnicki 2001b; Bitinaite et al. 2002; Pingoud et al. 2002). This was especially disappointing with respect to attempts to increase the length of the recognition site. Why was this hunt for longer recognition sites so important? The answer was their potential applications in gene therapy! To create a single unique dsDNA break within a mammalian genome, the REase would need to recognize DNA sequences of 16 bp or more (occurrence once every 416 bp = 4.3 × 109 bp). Expectations were high with the discovery of FokI. This R-M system from Flavobacterium okeanokoites was cloned in 1989, had separate DNA recognition and cleavage domains, required dimerization to produce a double-strand break, and cleaved 9/13 nucleotides downstream from the recognition site (Sugisaki and Kanazawa 1981; Kita et al. 1989; Looney et al. 1989; Kandavelou et al. 2004). Could one create a chimeric nuclease by fusing the nonspecific cleavage domain of FokI to zinc finger (ZF) domains to obtain a ZF nuclease (ZFN)? At the time, the structure of ZF domains made them the most versatile recognition motifs for the development of such artificial DNA-binding proteins (Pabo et al. 2001; Beerli and Barbas 2002; Kandavelou et al. 2004). Each ZF would bind a 3-bp DNA sequence, and tandem ZF motifs could increase the length of the sequence recognized (Kandavelou et al. 2004). FokI/3ZF would recognize a 9-bp inverted site, hence an effective recognition site of 18 bp, which hopefully would cut only once in a mammalian genome and stimulate homologous recombination at that single unique site. This should be feasible, as several laboratories had already reported homologous recombination at the cleavage site by ZFN (Bibikova et al. 2001, 2002; Porteus and Baltimore 2003). Srinivasan Chandrasegaran and colleagues in Baltimore saw “a glimpse of potential future therapeutic applications of ZFN in modifying and rewiring the human genome itself” (Kandavelou et al. 2004). Could chimeric nucleases be the new molecular scissors for research in stem cells? Would this technique eventually make correction of a genetic defect feasible, especially in treating single-gene diseases? In a decade or two, would gene therapy become routine in a clinical setting? In 2004, these studies were still in their infancy as gathered from the summary of the data on homologous recombination in frog oocytes, fruit flies (very inefficient), gene targeting in murine embryonic stem cells, and studies with CCR5 (the HIVs chemokine receptor) and CFTR (involved in cystic fibrosis) (Kandavelou et al. 2004). There would be many more hurdles to overcome in the years ahead (see Durai et al. 2005; Kandavelou et al. 2005, 2009; Mani et al. 2005a,b; Wu et al. 2007; Kandavelou and Chandrasegaran 2009; Ramalingam et al. 2011 for further details), some of them now no longer of consequence because of the arrival of CRISPR–Cas technology (Chapter 8).


Initially identified only in enterobacteriaceae because of limited detection methods (Bickle 1993; Bickle and Krüger 1993; King and Murray 1994; Barcus and Murray 1995), whole-genome sequencing had by the end of the century revealed candidate Type I R-M systems to be as abundant as Type II (Table 1). Using ATP as energy source, the intriguing Type I “molecular motor” proteins required 1D translocation along the DNA from the sequence-specific DNA site to the site of nonspecific cleavage (Murray 2000; Rao et al. 2000; Dryden et al. 2001; Bourniquel and Bickle 2002). Rather than biological tests, high sequence identity of putative HsdR and HsdM polypeptides allowed assignment of these to the Type IA, IB, IC, and ID subclasses, with 80%–99% identity even from different species (Barcus and Murray 1995). Between subclasses, identity was only ∼20%–35%, irrespective of the host. Despite this apparent lack of common ancestry, early coevolution was likely because of the unusual diversity of putative genes in distantly related species with intermediate levels of identity and the unlikelihood that Type I R-M systems would have evolved more than once (Sharp et al. 1992; Barcus and Murray 1995). This was reminiscent of other genes that discriminate “self” from “non-self” (e.g., the mammalian major histocompatibility complex), which led to the concept of “primitive bacterial immune system” (Barcus and Murray 1995).

Modeling the DNA Recognition Complex of the Type I M·EcoKI Trimeric Complex

Work on the structure of Type I and III REases was severely frustrated by the inability to generate crystals at that time. Undeterred, David Dryden and coworkers used 3D structures and folds of Type II MTases to build a picture of the structural domains of the trimeric M·EcoKI (M2S1) (Fig. 10).


FIGURE 10. Models of EcoKI and of the structure of the EcoKI restriction complex. The two TRDs of the specificity subunit HsdS (green) recognize the two halves of the DNA recognition site AAC(N6)GTGC. The TRDs are linked by conserved sequence regions that function as subunit interfaces and also define the length of the nonspecific DNA sequence in the middle of the recognition site. Two HsdM modification subunits (blue) bind to the conserved regions of HsdS via their amino- and carboxy-terminal domains. They wrap around the DNA helix on the opposite side of HsdS, allowing access of the methyltransferase domain of HsdM to DNA, presumably using base flipping as described for Type II MTases. Two HsdR subunits (orange) associate with HsdM and HsdS via the carboxyl terminus. The central part of the protein is involved in translocation and contains “DEAD box” motifs, characteristic of helicases (H). These motifs probably fold into two domains (IA and 2A) to form a cleft through which the DNA would pass (resembling a “RecA-like” structure that may be common to all helicases/translocases). EcoKI belongs to helicase superfamily 2 (SF2), whose members are believed to guide the DNA via regions outside the IA and IIA domains toward the cleft involving interactions with the DNA backbone (and not the bases), in line with the function of EcoKI as a DNA translocase rather than helicase. In the amino terminus of HsdR is a motif “X” characteristic of endonucleases (R), which is the PD…(D/E)XK common core (Figs. 6 and 7). The enzyme binds the target site via HsdM and HsdS using SAM as cofactor for binding and distinguishing between hemimethylated and unmodified DNA. If unmodified, the enzyme undergoes a large conformational change and translocates the DNA past itself, while remaining bound to the recognition site, creating large loops visible by EM and AFM, concomitant with ATP hydrolysis. The model rests on extensive genetic, biochemical, and biophysical evidence (see text for further details and references). (Inset A) A model of amino acids 43–157 from the amino-terminal TRD of EcoKI interaction with DNA (Sturrock and Dryden 1997). (Inset B) A front view from a partial model of a Type I MTase bound to DNA constructed using two copies of the structure of Type II MTases bound to DNA. The TRD regions are based on the structure of the TRD from M·HhaI and the methyltransferase domains in the catalytic domains of M·TaqI. Space filling shows sites of mutations resulting in loss of specificity and activity. (Inset C) Section of the HsdR subunit showing mutational analysis of conserved endonuclease and “DEAD box” (helicase-like) motifs. (Reprinted from Loenen 2003; originally adapted from Davies et al. 1999, with permission from Elsevier; A, from O'Neill et al. 1998, reproduced with permission from EMBO; also see Sturrock and Dryden 1997; B, reprinted from Dryden et al. 1995, with permission from Springer Nature; C, reprinted from Davies et al. 1999a,b, with permission from Elsevier.)


This DNA recognition/modification unit of the pentameric EcoKI complex required SAM for binding and distinguished hemimethylated (m6A-modified) DNA from unmodified DNA (Dryden et al. 1993; Powell and Murray 1995). Mutational analysis plus sequence comparisons and tertiary structure modeling indicated six motifs in the HsdM subunit common to the gamma class of Type II MTases (Cooper and Dryden 1994; Willcock et al. 1994; Dryden et al. 1995; Sturrock and Dryden 1997). This suggested a primordial MTase gene for Type I and Type II enzymes. In the model, two HsdM subunits (linked by the HsdS subunit) clamped the DNA, resembling two Type II MTases stacked together. Partial proteolysis indicated interaction of the carboxyl terminus of HsdR with HsdS (Davies et al. 1999b).

Mutational analysis, combined with alignment of 51 Type I HsdS TRD sequences in the database, and secondary structure predictions led to a tentative tertiary structure resembling that of M·HhaI (O'Neill et al. 1998). Each TRD would fit into the major groove and recognize the DNA, with the HsdM subunits arranged on either side of HsdS, allowing them to encircle the DNA and methylate the target adenines. Did this indicate that the MTases derived from a common ancestor with one monomeric TRD and a separate catalytic subunit (like some Type II MTases, e.g., AquI [Pinarbasi et al. 2003; Roberts et al. 2003b])? Two additional HsdR subunits would be responsible for bidirectional translocation and cleavage although complexes with a single HsdR could translocate DNA (Dryden et al. 1997; Janscak et al. 1998; Firman and Szczelkun 2000). This model was supported by other extensive experimental data, including DNA footprinting, fluorescence anisotropy, gel retardation, protein–DNA cross-linking, and measurements of the hydrodynamic shape of wild-type protein and mutants (Dryden et al. 1993, 1995; Powell et al. 1993, 1998a,b, 2003; Cooper and Dryden 1994; Willcock et al. 1994; Chen et al. 1995; Powell and Murray 1995; Sturrock and Dryden 1997; O'Neill et al. 1998, 2001).

The Molecular Motors of Type I and Type III REases

In addition to the above studies, which generated support for common ancestry of Type I and II DNA recognition domains and MTase functions, progress was made with respect to the curious translocation and restriction properties of the Type I and III REases. Multiple sequence alignments and structure predictions indicated an amino-terminal conserved motif “X” in the HsdR subunit of EcoKI, which was also present in other Type I and Type III enzymes and resembled the PD ··· (D/E)XK motif in Type II REases, described previously (Titheradge et al. 1996). This “X” motif is the active site, as “X” mutants could no longer restrict DNA but retained the ability to hydrolyze ATP and translocate DNA in vivo, thus uncoupling translocation and restriction (Davies et al. 1999a,b; Janscak et al. 1999b, 2001; Wang et al. 2000; Chang and Julin 2001). Similarly, “X” mutations in the carboxyl terminus of the Res subunit of EcoP15I also abolished DNA cleavage without affecting ATP hydrolysis (Janscak et al. 2001).

The putative translocation domain of HsdR of EcoKI and Res of EcoP15I shared so-called “DEAD box” (or helicase) motifs with proteins involved in replication, recombination, transcription, and repair that could unwind DNA, backtrack disrupted replication forks, remodel chromatin, or remove stalled RNA polymerase duplexes (West 1996; Park et al. 2002; Maluf et al. 2003; Whitehouse et al. 2003). This included DNA helicases and related AAA+ ATPases found in a wide variety of proteins from bacteria to humans. Did Type I and III REases share this functionality (West 1996)? The “DEAD box” motifs folded into a so-called RecA-like fold with a large cleft, through which the DNA could be either pushed or pulled (Gorbalenya and Koonin 1991; Murray et al. 1993; Titheradge et al. 1996; Aravind et al. 1999, 2000; Davies et al. 1999a,b; Caruthers and McKay 2002; Singleton and Wigley 2002, 2003). Sequence alignments and secondary structure predictions indicated that Type I REases belonged to the superfamily 2 (SF2) of the helicases (Gorbalenya and Koonin 1991; Murray et al. 1993; Titheradge et al. 1996; Hall and Matson 1999). This would be in line with mounting evidence that SF2 members often translocated or remodeled DNA without opening up the double helix (unlike many known members of the SF1, SF3, and SF4 superfamilies that did unwind DNA [Singleton and Wigley 2002]). It was likely that translocation by Type I REases would proceed via DNA–backbone interactions without strand separation or recognition of specific bases.

The crystal structures of several helicases revealed an ATP pocket consisting of the so-called “Walker” A and B boxes (helicase motifs I and II) first identified in ATP synthase (Walker et al. 1982) and an additional component Motif VI (Yao et al. 1997; Theis et al. 1999; Caruthers et al. 2000; Putnam et al. 2001; Caruthers and McKay 2002; Singleton and Wigley 2002; McClelland and Szczelkun 2004). These three motifs were strongly conserved in HsdR and Res (McClelland and Szczelkun 2004). Mutations in the “DEAD box” motifs in HsdR of EcoKI and Res of EcoP1I confirmed their importance in ATPase and endonuclease activity (Gorbalenya and Koonin 1991; Webb et al. 1996; Saha and Rao 1997; Davies et al. 1998, 1999a; Saha et al. 1998; Hall and Matson 1999; Singleton and Wigley 2002, 2003). Mutations in Walker A and B of EcoKI affected ATP binding and ATP hydrolysis, as expected; those in the other motifs provided the first formal proof for their involvement in translocation. Evidence for coupling of ATP hydrolysis to translocation was obtained using purified EcoKI mutant proteins: It could not linearize supercoiled DNA, had negligible ATPase activity in vitro, and also failed to translocate DNA in vivo. These latter results deserve particular mention, because of the novel use of EcoKI-mediated transfer of T7 DNA from the phage head into the cell (Fig. 11; Davies et al. 1999a; Garcia and Molineux 1999). EcoKI could pull the entire T7 chromosome (∼39 kb) into E. coli at ∼100–200 bp/sec (Davies et al. 1999a; Garcia and Molineux 1999), similar to the rates obtained in vitro with EcoKI and EcoR124I (Studier and Bandyopadhyay 1988; Firman and Szczelkun 2000).


FIGURE 11. In vivo translocation assay of EcoKI. A single target for EcoKI provides the means of bringing the T7 genome into an EcoKI-restricting cell (Davies et al. 1999a). (A) Infection of the cell commences with insertion of the first 1000 bp of the T7 genome, which carries one unmodified recognition site for EcoKI. (B) Normal entry of T7 DNA is mediated by RNA polymerases, both E. coli and T7, which can be blocked by rifampicin and chloramphenicol. DNA translocation by EcoKI bound to its unmodified target site substitutes for RNA polymerase and pulls in the DNA from the phage head. (C) E. coli Dam methylates GATC sites that enter the cell. The fraction of DNA that has entered the cell can thus be estimated by comparing digests with the methylation-sensitive DpnI and the methylation-insensitive Sau3A. The entire T7 chromosome (∼39 kb) can be pulled into the cell by EcoKI and the rate of entry was calculated to be 100–200 bp/sec (Davies et al. 1999a; Garcia and Molineux 1999), a figure similar to those (200–400 bp/sec) obtained from in vitro experiments with EcoKI and EcoR124I (Studier and Bandyopadhyay 1988; Firman and Szczelkun 2000). (Note that for technical reasons a mutant T7 0.3 phage was used, which will be explained elsewhere.) (Reprinted with permission of Microbiology Society from Murray 2002.)


Mark Szczelkun compared nearly 200 characterized and putative open reading frames (ORFs) encoding HsdR and Res subunits in Type I and III systems, respectively, which confirmed the conservation of the “X” and “DEAD box” motifs (Gorbalenya and Koonin 1991; Murray et al. 1993; Titheradge et al. 1996; Davies et al. 1999b; Janscak et al. 1999b, 2001; McClelland and Szczelkun 2004). In HsdR, “X” was always ahead of the motor, in Res, behind it, in line with the long-held assumption that HsdR cut distant to the recognition site, and Res proximal. The comparison revealed a new putative helicase motif, the Q-tip helix (McClelland and Szczelkun 2004), also found in PcrA, rep, UvrB, RecG, and RuvB, and implicated in the activity of DEAD-box RNA helicases, RuvB, and BLM (Subramanya et al. 1996; Korolev et al. 1997; Bähr et al. 1998; Theis et al. 1999; Velankar et al. 1999; Iwasaki et al. 2000; Putnam et al. 2001; Singleton et al. 2001; Tanner et al. 2003). The importance of the Q-tip in Type I and III REases remained to be established. Outside these regions, homology was low and polypeptides sometimes lacked amino or carboxyl termini. The 39 Type III Res sequences split into two groups, IIIA and IIIB. The IIIA sequences were closely related, whereas the IIIB sequences were less well conserved, and, intriguingly, resembled HsdR more than IIIA (Titheradge et al. 1996; Davies et al. 1998, 1999a). However, both IIIA and IIIB REases cleaved DNA typical of Type III enzymes, and not Type I, perhaps indicative of a gradual transition between Type I, IIIA, and IIIB (McClelland and Szczelkun 2004).

HsdR or Res had no activity on their own, like many other superfamily members (Cooper and Dryden 1994; Dryden et al. 1997, 2001; Sturrock and Dryden 1997; O'Neill et al. 1998; Murray 2000; Rao et al. 2000; Szczelkun 2000; Bourniquel and Bickle 2002; Delagoutte and von Hippel 2003). Were one or two HsdR subunits used or needed? This reflected the debate over whether superfamily members act as monomers or dimers in their respective complexes (Dryden et al. 1997; Janscak et al. 1998; Firman and Szczelkun 2000; Nanduri et al. 2002; Maluf et al. 2003). EcoKI probably could translocate bidirectionally in vivo as well as in vitro, but EcoBI apparently could not, at least not in vitro (Powell et al. 1998b). Were the HsdR subunits acting independently of each other? This was a puzzle, because translocation studies with a single HsdR suggested that when a subunit released DNA during movement, it could bind and translocate the DNA on the opposite side of the complex (Firman and Szczelkun 2000; McClelland and Szczelkun 2004). Did perhaps nonspecific DNA wrap around the complex and stimulate translocation and/or cleavage (Mernagh and Kneale 1996; Szczelkun et al. 1996; McClelland and Szczelkun 2004)? It was too early to say (Szczelkun et al. 1996; Janscak et al. 1999a). Translocation by HsdR might involve contacting nonspecific DNA adjacent to the recognition site in a cleft, which would close and reopen, a process governed by ATP, Mg2+, and probably SAM, to fuel and control the conformational changes. This would be in line with observations of substantial movements and rearrangements of different domains after ATP hydrolysis, which would allow helicase or translocase activity, respectively (Korolev et al. 1997; Hall and Matson 1999; Velankar et al. 1999; Singleton et al. 2001; Singleton and Wigley 2002; Mahdi et al. 2003).

The debate on the choice of the cut site with respect to the recognition site had started in the 1980s with Bill Studier's collision model (Chapter 6; Studier and Bandyopadhyay 1988). Evidence for cooperation between sites was supported by additional in vivo experiments and in vitro by atomic force microscopy (AFM) (Webb et al. 1996; O'Neill et al. 1998; Ellis et al. 1999). This was good news, as AFM, in contrast to the harsh treatment used in the EM, allowed gentle sample preparation via noncovalent attachment of protein–DNA complexes to a mica surface in aqueous solution. These data showed more efficient cleavage of linear DNA with two sites than with one site (Ellis et al. 1999). Two EcoKI complexes apparently bound their respective recognition sites on opposite sides of the target plasmid, dimerized, looped the DNA, and cut it ∼7 min after addition of ATP, because of stalling of the complex upon excessive DNA supercoiling or maximal contraction of the DNA loop between the two bound EcoKI molecules. Interestingly, translocase mutants were still capable of dimerization, which might occur between any two occupied (not only adjacent) EcoKI sites, although interaction between adjacent sites was most probable (Ellis et al. 1999; Berge et al. 2000). This led to a variant of the collision model in which two EcoKI complexes could dimerize before translocation (Ellis et al. 1999). Collision with another protein or structure (e.g., a Holliday junction) would also stop translocation and result in DNA cleavage (Studier and Bandyopadhyay 1988; Janscak et al. 1999a; Murray 2000; Dryden et al. 2001).

Many questions remained: Would HsdR touch one strand or both strands of the dsDNA via backbone contacts; and what about the step size or amount of DNA transported per physical step? SF1 helicases stepped anything from 1–2 bp, 3–5 bp, or even 23 bp (Roman and Kowalczykowski 1989; Ali and Lohman 1997; Bianco and Kowalczykowski 2000; Dillingham et al. 2000; Kim et al. 2002). And why would no cleavage occur during initial translocation? Was the translocation rate too high or was the “X” site in the wrong conformation to contact the DNA? Easier to answer was the question about the nature of the DNA ends at the cut site: sticky or blunt end? This proved to depend on the REase: EcoKI (Type IA), EcoAI (Type IB), and EcoR124I (Type IC) cut randomly without preference for particular sequences, with 5′ and 3′ overhangs of varying length (Jindrova et al. 2005). The final conclusion was that two REases were needed for DSBs, each one providing one catalytic center for cleavage of one strand (Jindrova et al. 2005).

As mentioned in the Introduction, research on the Type III REases was limited to the enzymes from phage P1 and plasmid P15. It was unclear why EcoP1I and EcoP15I needed a second Mod subunit in the MTase, as methylation occurred on only one strand of the recognition sequence (Humbelin et al. 1988; Ahmad et al. 1995). The Mod subunits dictated specific recognition and methylation and the Res subunits translocation and cutting. In the case of EcoP15I, Res2Mod2 (again, why two of each subunit?) acted as MTase in the absence of ATP and as either MTase or REase in the presence of ATP, depending on the methylation state of the recognition site (Janscak et al. 2001). Did the second Mod subunit perhaps stabilize the complex via nonspecific DNA binding, similar to the Type IIS REase BspMI (Gormley et al. 2002)?

Although DNA translocation had been proven unambiguously for Type I enzymes, there was no convincing evidence for a similar mechanism for Type III enzymes (Murray 2000; Rao et al. 2000; Szczelkun 2000; Dryden et al. 2001; Bourniquel and Bickle 2002). Based on the Type I model, cleavage would occur after DNA tracking using ATP as the energy source and collision by two Res2Mod2 complexes, which remained attached to their two head-to-head recognition sites via the MTase part of Res2Mod2 (Fig. 12; Krüger et al. 1995; Meisel et al. 1995; Saha and Rao 1995; Rao et al. 2000; Dryden et al. 2001; Bourniquel and Bickle 2002). Collision would result in a conformational change and cleavage 25–27 bp downstream from one of the two recognition sites. This would destabilize the complex, preventing restriction of the second site (Janscak et al. 2001). Uncertainty remained as to whether translocation occurred in one or both directions and whether perhaps two Res subunits cooperated to translocate DNA unidirectionally (McClelland and Szczelkun 2004).


FIGURE 12. DNA tracking collision model for Type III REases (one Mod and Res subunit are shown for clarity [Meisel et al. 1995]). A pair of head-to-head–oriented recognition sites (→) is occupied by one enzyme molecule each. Mod is shown in blue and Res in red. Both enzyme-site complexes use ATP to translocate DNA (Meisel et al. 1995; Saha and Rao 1995), shown by a loop of increasing size, and convergently track until they collide. The resulting collision complex elicits a conformational change and results in cleavage of the DNA 25–27 bp downstream from one of the two recognition sites. This destabilizes the complex, preventing restriction of the second site (Janscak et al. 2001). (Adapted from Meisel et al. 1995, with permission from EMBO.)


Restriction Alleviation of Type I REases by ClpXP

Both early gene transfer experiments and later studies supported the notion that Type I genes easily replaced alleles with different recognition sites or, alternatively, mutant genes encoding nonmodifying proteins (Arber 1965; Arber and Linn 1969; Prakash-Cheng and Ryu 1993; Prakash-Cheng et al. 1993; Naito et al. 1995; Kobayashi 1996, 1998, 2001; O'Neill et al. 1997; Nakayama and Kobayashi 1998; Hurst and Werren 2001). This indicated that the incoming REase did not destroy the host DNA, despite the absence of cognate methylation on the chromosome. What was the mechanism behind this restriction alleviation (RA)? Did it perhaps involve subunit (dis)assembly (Dryden et al. 1997; Janscak et al. 1998)? Or was there perhaps another, general, protective mechanism to protect unmodified sites on the host DNA upon DNA damage, DNA repair, transfer of hsd genes, and/or recombination (Bertani and Weigle 1953; Makovets et al. 1998, 1999; Murray 2000, 2002; Doronina and Murray 2001)? Both in vivo and in vitro experiments helped to resolve this important issue. Complementation between lambda hsdK phages and host mutants, as well as western blots led to the discovery of intricate posttranslational control of the HsdR subunit of EcoKI by the ATP-dependent ClpXP protease (Makovets et al. 1998, 1999; Doronina and Murray 2001). Surprisingly, use of HsdR and HsdM mutants showed that the HsdR subunit was degraded by ClpXP when modification was impaired but only after assembly of a specific DNA/EcoKI-translocation-proficient complex. In other words, ClpXP would degrade HsdR during translocation, but not before (Fig. 13). In line with this, “DEAD box” mutants were resistant to degradation, whereas “X” mutants would be degraded like wild-type enzyme. Western blots showed this ClpXP-dependent degradation to be solely aimed at HsdR, whereas HsdM remained intact.


FIGURE 13. Model for the mechanism of ClpXP-dependent proteolytic control of restriction by EcoKI. ATP-dependent translocation of DNA by EcoKI occurs after the enzyme binds unmodified recognition sequences on the host chromosome. However, ClpXP recognizes HsdR during translocation and destroys the restriction subunits of the EcoKI complex (but leaves the trimeric methylase intact), thereby preventing further translocation and cutting of the chromosome. (Reprinted from Murray 2000, with permission from American Society for Microbiology; see also Loenen 2003.)


This extraordinary control of restriction to prevent chromosome degradation, if modification became even temporarily insufficient, was also active against EcoAI but not EcoR124I (Makovets et al. 1998, 1999; Doronina and Murray 2001). Although foreign (phage) DNA was destroyed by a reconstituted restriction-proficient methyltransfer-deficient EcoKI complex, all (600-odd) chromosomal target sequences remained unharmed, which led to the concept of “self” and “non-self” (Murray 2002). This was in contrast with Type II REases, which did cut the host DNA when modification of the host DNA became inadequate. These REases maintained themselves by cleaving non-self DNA, and therefore Ichizo Kobayashi called these enzymes “selfish” (Kobayashi 2001). The experiments with EcoKI and other Type I systems by Noreen Murray and colleagues clearly indicated otherwise (O'Neill et al. 1997; Murray 2002). The “pro selfish” camp stated that the REase would not only destroy incoming foreign DNA but also enhance the frequency of horizontal transfer of R-M systems into other genomes by generating recombinogenic free DNA ends in the cell (Jeltsch and Pingoud 1996; Kobayashi 2001), somewhat similar to homing endonucleases (Gimble 2000). The proposed role for selfish and non-selfish REases need not be contradictive. DNA translocation of R-M systems could aid incorporation of unmodified DNA into the recipient chromosome after conjugation or transduction of large chunks of incoming DNA. Random cleavage by Type I enzymes into smaller fragments might generate suitable DNA substrates for the host's major recombination complex RecBC at Chi sites, and benefit populations as well as individual cells (Price and Bickle 1986; Barcus and Murray 1995; Murray 2002). In other words, although not essential, R-M systems might influence the stability of chromosomes at the population level, which must be neither too static nor too fluid, allowing influx of foreign DNA to enhance survival of the population when conditions change, leading to mosaic sequences and slow evolution toward new species (Price and Bickle 1986; Wilkins 2000; Murray 2002; Arber 2003). The role of translocation by Type I enzymes in this level of maintenance of chromosome integrity and evolution and dissection of RA pathways by bacterial hosts and their enemies would await further exploration (Murray 2002).


The Seventh NEB Meeting on DNA Restriction and Modification August 24–29, 2015. Uniwersytet Gdański ulica, Kladki 24, Gdańsk, Poland.

Adzuma K, Mizuuchi K. 1989. Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction. Cell 57: 41–47. 10.1016/0092-8674(89)90170-0

Aggarwal AK. 1995. Structure and function of restriction endonucleases. Curr Opin Struct Biol 5: 11–19. 10.1016/0959-440X(95)80004-K

Ahmad I, Krishnamurthy V, Rao DN. 1995. DNA recognition by the EcoP15I and EcoPI modification methyltransferases. Gene 157: 143–147. 10.1016/0378-1119(95)00671-R

Ali JA, Lohman TM. 1997. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275: 377–380. 10.1126/science.275.5298.377

Alves J, Vennekohl P. 2004. Protein engineering of restriction enzymes. In Restriction endonucleases (ed. Pingoud A), pp. 393–411. Springer, Berlin.

Alves J, Rüter T, Geiger R, Fliess A, Maass G, Pingoud A. 1989. Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC. Biochemistry 28: 2678–2684. 10.1021/bi00432a047

Anderson JE. 1993. Restriction endonucleases and modification methylases. Curr Opin Struct Biol 3: 24–30. 10.1016/0959-440X(93)90197-S

Anton BP, Heiter DF, Benner JS, Hess EJ, Greenough L, Moran LS, Slatko BE, Brooks JE. 1997. Cloning and characterization of the BglII restriction-modification system reveals a possible evolutionary footprint. Gene 187: 19–27. 10.1016/S0378-1119(96)00638-5

Aravind L, Makarova KS, Koonin EV. 2000. SURVEY AND SUMMARY: Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. Nucleic Acids Res 28: 3417–3432. 10.1093/nar/28.18.3417

Aravind L, Walker DR, Koonin EV. 1999. Conserved domains in DNA repair proteins and evolution of repair systems. Nucleic Acids Res 27: 1223–1242. 10.1093/nar/27.5.1223

Arber W. 1965. Host-controlled modification of bacteriophage. Ann Rev Microbiol 19: 365–378. 10.1146/annurev.mi.19.100165.002053

Arber W. 1966. Host specificity of DNA produced by Escherichia coli. 9. Host-controlled modification of bacteriophage fd. J Mol Biol 20: 483–496. 10.1016/0022-2836(66)90004-0

Arber W. 1979. Promotion and limitation of genetic exchange. Science 205: 361–365. 10.1126/science.377489

Arber W. 2000. Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS Microbiol Rev 24: 1–7. 10.1111/j.1574-6976.2000.tb00529.x

Arber W. 2003. Elements for a theory of molecular evolution. Gene 317: 3–11. 10.1016/S0378-1119(03)00654-1

Arber W, Linn S. 1969. DNA modification and restriction. Ann Rev Biochem 38: 467–500. 10.1146/annurev.bi.38.070169.002343

Arber W, Morse ML. 1965. Host specificity of DNA produced by Escherichia coli. VI. Effects on bacterial conjugation. Genetics 51: 137–148.

Athanasiadis A, Vlassi M, Kotsifaki D, Tucker PA, Wilson KS, Kokkinidis M. 1994. Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV. Nat Struct Biol 1: 469–475. 10.1038/nsb0794-469

Bähr A, De Graeve F, Kedinger C, Chatton B. 1998. Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein. Oncogene 17: 2565–2571. 10.1038/sj.onc.1202389

Baldwin GS, Vipond IB, Halford SE. 1995. Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease. Biochemistry 34: 705–714. 10.1021/bi00002a038

Ban C, Yang W. 1998. Structural basis for MutH activation in E. coli mismatch repair and relationship of MutH to restriction endonucleases. EMBO J 17: 1526–1534. 10.1093/emboj/17.5.1526

Bannister D, Glover SW. 1968. Restriction and modification of bacteriophages by R+ strains of Escherichia coli K12. Biochem Biophys Res Commun 30: 735–738. 10.1016/0006-291X(68)90575-5

Bannister D, Glover SW. 1970. The isolation and properties of non-restricting mutants of two different host specificities associated with drug resistance factors. J Gen Microbiol 61: 63–71. 10.1099/00221287-61-1-63

Barcus VA, Murray NE. 1995. Barriers to recombination: restriction. Population genetics of bacteria (ed. Baumberg S, Young JPW, Saunders SR, Wellington EMH), pp. 31–58. Society for General Microbiology, Cambridge University Press, Cambridge.

Bath AJ, Milsom SE, Gormley NA, Halford SE. 2002. Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA. J Biol Chem 277: 4024–4033. 10.1074/jbc.M108441200

Baxter BK, Topal MD. 1993. Formation of a cleavasome: enhancer DNA-2 stabilizes an active conformation of NaeI dimer. Biochemistry 32: 8291–8298. 10.1021/bi00083a033

Beerli RR, Barbas CF. 2002. Engineering polydactyl zinc-finger transcription factors. Nat Biotech 20: 135–141. 10.1038/nbt0202-135

Beese LS, Steitz TA. 1991. Structural basis for the 3′–5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J 10: 25–33. 10.1002/j.1460-2075.1991.tb07917.x

Bennett SP, Halford SE. 1989. Recognition of DNA by type II restriction enzymes. Curr Top Cell Regul 30: 57–104. 10.1016/B978-0-12-152830-0.50005-0

Berge T, Ellis DJ, Dryden DT, Edwardson JM, Henderson RM. 2000. Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy. Biophys J 79: 479–484. 10.1016/S0006-3495(00)76309-0

Bertani G, Weigle JJ. 1953. Host controlled variation in bacterial viruses. J Bacteriol 65: 113–121.

Bianco PR, Kowalczykowski SC. 2000. Translocation step size and mechanism of the RecBC DNA helicase. Nature 405: 368–372. 10.1038/35012652

Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S. 2001. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21: 289–297. 10.1128/MCB.21.1.289-297.2001

Bibikova M, Golic M, Golic KG, Carroll D. 2002. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161: 1169–1175.

Bickle TA. 1993. The ATP-dependent restriction enzymes. In Nucleases, 2nd ed. (ed. Linn SM, Lloyd SR, Roberts RJ), pp. 88–109. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Bickle TA, Krüger DH. 1993. Biology of DNA restriction. Microbiol Rev 57: 434–450.

Bigger CH, Murray K, Murray NE. 1973. Recognition sequence of a restriction enzyme. Nature: New Biol 244: 7–10.

Bilcock DT, Halford SE. 1999. DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli. Mol Microbiol 31: 1243–1254. 10.1046/j.1365-2958.1999.01266.x

Bilcock DT, Daniels LE, Bath AJ, Halford SE. 1999. Reactions of type II restriction endonucleases with 8-base pair recognition sites. J Biol Chem 274: 36379–36386. 10.1074/jbc.274.51.36379

Bitinaite J, Schildkraut I. 2002. Self-generated DNA termini relax the specificity of SgrAI restriction endonuclease. Proc Natl Acad Sci 99: 1164–1169. 10.1073/pnas.022346799

Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I. 1998. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci 95: 10570–10575. 10.1073/pnas.95.18.10570

Bitinaite J, Mitkaite G, Dauksaite V, Jakubauskas A, Timinskas A, Vaisvila R, Lubys A, Janulaitis A. 2002. Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences. Mol Genet Genomics 267: 664–672. 10.1007/s00438-002-0701-6

Black CB, Cowan JA. 1994. Inorg Chem 33: 5805–5808. 10.1021/ic00103a030

Blattler MO, Wenz C., Pingoud A, Benner SA. 1998. Distorting duplex DNA by dimethylenesulfone substitution: a new class of ‘transition state analog’ inhibitors of restriction enzymes. J Am Chem Soc 120: 2674–2675. 10.1021/ja972768y

Blumenthal RM, Cheng X. 2001. A Taq attack displaces bases. Nat Struct Biol 8: 101–103. 10.1038/84072

Bochtler M, Szczepanowski RH, Tamulaitis G, Grazulis S, Czapinska H, Manakova E, Siksnys V. 2006. Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO J 25: 2219–2229. 10.1038/sj.emboj.7601096

Bourniquel AA, Bickle TA. 2002. Complex restriction enzymes: NTP-driven molecular motors. Biochimie 84: 1047–1059. 10.1016/S0300-9084(02)00020-2

Boyer HW, Chow LT, Dugaiczyk A, Hedgpeth J, Goodman HM. 1973. DNA substrate site for the EcoRII restriction endonuclease and modification methylase. Nature: New Biol 244: 40–43. 10.1038/244040a0

Bozic D, Grazulis S, Siksnys V, Huber R. 1996. Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 A resolution. J Mol Biol 255: 176–186. 10.1006/jmbi.1996.0015

Brooks JE, Nathan PD, Landry D, Sznyter LA, Waite-Rees P, Ives CL, Moran LS, Slatko BE, Benner JS. 1991. Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts. Nucleic Acids Res 19: 841–850. 10.1093/nar/19.4.841

Bujnicki JM. 2000a. Homology modelling of the DNA 5mC methyltransferase M.BssHII. Is permutation of functional subdomains common to all subfamilies of DNA methyltransferases? Int J Biol Macromol 27: 195–204. 10.1016/S0141-8130(00)00120-3

Bujnicki JM. 2000b. Phylogeny of the restriction endonuclease-like superfamily inferred from comparison of protein structures. J Mol Evol 50: 39–44. 10.1007/s002399910005

Bujnicki JM. 2001a. A model of structure and action of Sau3AI restriction endonuclease that comprises two MutH-like endonuclease domains within a single polypeptide. Acta Microbiol Pol 50: 219–231.

Bujnicki JM. 2001b. Understanding the evolution of restriction-modification systems: clues from sequence and structure comparisons. Acta Biochim Pol 48: 935–967.

Bujnicki JM. 2003. Crystallographic and bioinformatic studies on restriction endonucleases: inference of evolutionary relationships in the “midnight zone” of homology. Curr Protein Pept Sci 4: 327–337. 10.2174/1389203033487072

Bujnicki JM. 2004. Molecular phylogenetics of restriction endonucleases. In Restriction endonucleases (ed. Pingoud A), pp. 63–93. Springer, Berlin.

Bujnicki JM, Rychlewski L. 2001a. Grouping together highly diverged PD-(D/E)XK nucleases and identification of novel superfamily members using structure-guided alignment of sequence profiles. J Mol Microbiol Biotechnol 3: 69–72.

Bujnicki JM, Rychlewski L. 2001b. The herpesvirus alkaline exonuclease belongs to the restriction endonuclease PD-(D/E)XK superfamily: insight from molecular modeling and phylogenetic analysis. Virus Genes 22: 219–230. 10.1023/A:1008131810233

Bujnicki JM, Radlinska M, Rychlewski L. 2001. Polyphyletic evolution of type II restriction enzymes revisited: two independent sources of second-hand folds revealed. Trends Biochem Sci 26: 9–11. 10.1016/S0968-0004(00)01690-X

Carlson K, Kosturko LD. 1998. Endonuclease II of coliphage T4: a recombinase disguised as a restriction endonuclease? Mol Microbiol 27: 671–676. 10.1046/j.1365-2958.1998.00728.x

Carrick KL, Topal MD. 2003. Amino acid substitutions at position 43 of NaeI endonuclease. Evidence for changes in NaeI structure. J Biol Chem 278: 9733–9739. 10.1074/jbc.M209192200

Caruthers JM, McKay DB. 2002. Helicase structure and mechanism. Curr Opin Struct Biol 12: 123–133. 10.1016/S0959-440X(02)00298-1

Caruthers JM, Johnson ER, McKay DB. 2000. Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Proc Natl Acad Sci 97: 13080–13085. 10.1073/pnas.97.24.13080

Cech D, Pein CD, Kubareva EA, Gromova ES, Oretskaya ES, Shabarova ZA. 1988. Influence of modifications on the cleavage of oligonucleotide duplexes by EcoRII and MvaI endonucleases. Nucleosides Nucleotides 7: 585–588. 10.1080/07328318808056290

Chang HW, Julin DA. 2001. Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family. J Biol Chem 276: 46004–46010. 10.1074/jbc.M108627200

Chen A, Powell LM, Dryden DT, Murray NE, Brown T. 1995. Tyrosine 27 of the specificity polypeptide of EcoKI can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence. Nucleic Acids Res 23: 1177–1183. 10.1093/nar/23.7.1177

Cheng X, Blumenthal RM. 2002. Cytosines do it, thymines do it, even pseudouridines do it—base flipping by an enzyme that acts on RNA. Structure 10: 127–129. 10.1016/S0969-2126(02)00710-4

Cheng X, Roberts RJ. 2001. AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res 29: 3784–3795. 10.1093/nar/29.18.3784

Cheng X, Kumar S, Klimasauskas S, Roberts RJ. 1993a. Crystal structure of the HhaI DNA methyltransferase. Cold Spring Harbor Symp Quant Biol 58: 331–338. 10.1101/SQB.1993.058.01.039

Cheng X, Kumar S, Posfai J, Pflugrath JW, Roberts RJ. 1993b. Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell 74: 299–307. 10.1016/0092-8674(93)90421-L

Cheng X, Balendiran K, Schildkraut I, Anderson JE. 1994. Structure of PvuII endonuclease with cognate DNA. EMBO J 13: 3927–3935. 10.1002/j.1460-2075.1994.tb06708.x

Chevalier BS, Monnat RJ Jr, Stoddard BL. 2001. The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites. Nat Struct Biol 8: 312–316. 10.1038/86181

Chinen A, Uchiyama I, Kobayashi I. 2000. Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms. Gene 259: 109–121. 10.1016/S0378-1119(00)00459-5

Choi J. 1994. “Crystal structure analysis of site-directed mutants of EcoRI endonuclease complexed to DNA.” PhD thesis, University of Pittsburgh.

Colandene JD, Topal MD. 1998. The domain organization of NaeI endonuclease: separation of binding and catalysis. Proc Natl Acad Sci 95: 3531–3536. 10.1073/pnas.95.7.3531

Conlan LH, Dupureur CM. 2002a. Dissecting the metal ion dependence of DNA binding by PvuII endonuclease. Biochemistry 41: 1335–1342. 10.1021/bi015843x

Conlan LH, Dupureur CM. 2002b. Multiple metal ions drive DNA association by PvuII endonuclease. Biochemistry 41: 14848–14855. 10.1021/bi026403o

Conlan LH, Jose TJ, Thornton KC, Dupureur CM. 1999. Modulating restriction endonuclease activities and specificities using neutral detergents. BioTechniques 27: 955–960. 10.2144/99275st02

Connolly BA, Eckstein F, Pingoud A. 1984. The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction. J Biol Chem 259: 10760–10763.

Connolly BA, Liu HH, Parry D, Engler LE, Kurpiewski MR, Jen-Jacobson L. 2001. Assay of restriction endonucleases using oligonucleotides. Methods Mol Biol 148: 465–490.

Conrad M, Topal MD. 1989. DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI. Proc Natl Acad Sci 86: 9707–9711. 10.1073/pnas.86.24.9707

Conrad M, Topal MD. 1992. Modified DNA fragments activate NaeI cleavage of refractory DNA sites. Nucleic Acids Res 20: 5127–5130. 10.1093/nar/20.19.5127

Cooper LP, Dryden DT. 1994. The domains of a type I DNA methyltransferase. Interactions and role in recognition of DNA methylation. J Mol Biol 236: 1011–1021. 10.1016/0022-2836(94)90008-6

Cowan JA. 1998. Metal activation of enzymes in nucleic acid biochemistry. Chem Rev 98: 1067–1088. 10.1021/cr960436q

Cowan JA. 2004. Role of metal ions in promoting DNA binding and cleavage by restriction endonucleases. In Restriction endonucleases (ed. Pingoud A), pp. 339–360. Springer, Berlin.

Davies GP, Powell LM, Webb JL, Cooper LP, Murray NE. 1998. EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities. Nucleic Acids Res 26: 4828–4836. 10.1093/nar/26.21.4828

Davies GP, Kemp P, Molineux IJ, Murray NE. 1999a. The DNA translocation and ATPase activities of restriction-deficient mutants of EcoKI. J Mol Biol 292: 787–796. 10.1006/jmbi.1999.3081

Davies GP, Martin I, Sturrock SS, Cronshaw A, Murray NE, Dryden DT. 1999b. On the structure and operation of type I DNA restriction enzymes. J Mol Biol 290: 565–579. 10.1006/jmbi.1999.2908

Deibert M, Grazulis S, Janulaitis A, Siksnys V, Huber R. 1999. Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 Å resolution. EMBO J 18: 5805–5816. 10.1093/emboj/18.21.5805

Deibert M, Grazulis S, Sasnauskas G, Siksnys V, Huber R. 2000. Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA. Nat Struct Biol 7: 792–799. 10.1038/79032

Delagoutte E, von Hippel PH. 2003. Helicase mechanisms and the coupling of helicases within macromolecular machines. Part II: integration of helicases into cellular processes. Q Rev Biophys 36: 1–69. 10.1017/S0033583502003864

Demple B, Johnson A, Fung D. 1986. Exonuclease III and endonuclease IV remove 3′ blocks from DNA synthesis primers in H2O2-damaged Escherichia coli. Proc Natl Acad Sci 83: 7731–7735. 10.1073/pnas.83.20.7731

Dillingham MS, Wigley DB, Webb MR. 2000. Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed. Biochemistry 39: 205–212. 10.1021/bi992105o

Dominguez MA Jr, Thornton KC, Melendez MG, Dupureur CM. 2001. Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease. Proteins 45: 55–61. 10.1002/prot.1123

Dorner LF, Schildkraut I. 1994. Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. Nucleic Acids Res 22: 1068–1074. 10.1093/nar/22.6.1068

Doronina VA, Murray NE. 2001. The proteolytic control of restriction activity in Escherichia coli K-12. Mol Microbiol 39: 416–428. 10.1046/j.1365-2958.2001.02232.x

Dryden DT. 2013. The architecture of restriction enzymes. Structure 21: 1720–1721. 10.1016/j.str.2013.09.009

Dryden DT, Cooper LP, Murray NE. 1993. Purification and characterization of the methyltransferase from the type 1 restriction and modification system of Escherichia coli K12. J Biol Chem 268: 13228–13236.

Dryden DT, Sturrock SS, Winter M. 1995. Structural modelling of a type I DNA methyltransferase. Nat Struct Biol 2: 632–635. 10.1038/nsb0895-632

Dryden DT, Cooper LP, Thorpe PH, Byron O. 1997. The in vitro assembly of the EcoKI type I DNA restriction/modification enzyme and its in vivo implications. Biochemistry 36: 1065–1076. 10.1021/bi9619435

Dryden DT, Murray NE, Rao DN. 2001. Nucleoside triphosphate-dependent restriction enzymes. Nucleic Acids Res 29: 3728–3741. 10.1093/nar/29.18.3728

Dunn TM, Hahn S, Ogden S, Schleif RF. 1984. An operator at –280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. Proc Natl Acad Sci 81: 5017–5020. 10.1073/pnas.81.16.5017

Dupureur CM, Conlan LH. 2000. A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions. Biochemistry 39: 10921–10927. 10.1021/bi000337d

Dupureur CM, Dominguez MA Jr, . 2001. The PD…(D/E)XK motif in restriction enzymes: a link between function and conformation. Biochemistry 40: 387–394. 10.1021/bi001680l

Dupureur CM, Hallman LM. 1999. Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases. Eur J Biochem/FEBS 261: 261–268. 10.1046/j.1432-1327.1999.00265.x

Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S. 2005. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33: 5978–5990. 10.1093/nar/gki912

Ellis DJ, Dryden DT, Berge T, Edwardson JM, Henderson RM. 1999. Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy. Nat Struct Biol 6: 15–17. 10.1038/4882

Embleton ML, Siksnys V, Halford SE. 2001. DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites. J Mol Biol 311: 503–514. 10.1006/jmbi.2001.4892

Engler LE. 1998. “Specificity determinants in the BamHI-endonuclease-DNA interaction.” PhD thesis, University of Pittsburgh.

Engler LE, Welch KK, Jen-Jacobson L. 1997. Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. J Mol Biol 269: 82–101. 10.1006/jmbi.1997.1027

Engler LE, Sapienza P, Dorner LF, Kucera R, Schildkraut I, Jen-Jacobson L. 2001. The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC. J Mol Biol 307: 619–636. 10.1006/jmbi.2000.4428

Erskine SG, Halford SE. 1998. Reactions of the eco RV restriction endonuclease with fluorescent oligodeoxynucleotides: identical equilibrium constants for binding to specific and non-specific DNA. J Mol Biol 275: 759–772. 10.1006/jmbi.1997.1517

Firman K, Szczelkun MD. 2000. Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement. EMBO J 19: 2094–2102. 10.1093/emboj/19.9.2094

Fliess A, Wolfes H, Seela F, Pingoud A. 1988. Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides. Nucleic Acids Res 16: 11781–11793. 10.1093/nar/16.24.11781

Flores H, Osuna J, Heitman J, Soberon X. 1995. Saturation mutagenesis of His114 of EcoRI reveals relaxed-specificity mutants. Gene 157: 295–301. 10.1016/0378-1119(94)00863-N

Friedhoff P, Kolmes B, Gimadutdinow O, Wende W, Krause KL, Pingoud A. 1996. Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res 24: 2632–2639. 10.1093/nar/24.14.2632

Friedhoff P, Lurz R, Luder G, Pingoud A. 2001. Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops. J Biol Chem 276: 23581–23588. 10.1074/jbc.M101694200

Fritz A, Kuster W, Alves J. 1998. Asn141 is essential for DNA recognition by EcoRI restriction endonuclease. FEBS Lett 438: 66–70. 10.1016/S0014-5793(98)01274-5

Fuxreiter M, Osman R. 2001. Probing the general base catalysis in the first step of BamHI action by computer simulations. Biochemistry 40: 15017–15023. 10.1021/bi010987x

Gabbara S, Bhagwat AS. 1992. Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites. J Biol Chem 267: 18623–18630.

Garcia LR, Molineux IJ. 1999. Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI. Proc Natl Acad Sci 96: 12430–12435. 10.1073/pnas.96.22.12430

Garcia RA, Bustamante CJ, Reich NO. 1996. Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA. Proc Natl Acad Sci 93: 7618–7622. 10.1073/pnas.93.15.7618

Geiger R, Rüter T, Alves J, Fliess A, Wolfes H, Pingoud V, Urbanke C, Maass G, Pingoud A, Dusterhoft A, et al. 1989. Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants. Biochemistry 28: 2667–2677. 10.1021/bi00432a046

Ghosh A, Passaris I, Tesfazgi Mebrhatu M, Rocha S, Vanoirbeek K, Hofkens J, Aertsen A. 2014. Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli. Nucleic Acids Res 42: 3908–3918. 10.1093/nar/gkt1370

Gimble FS. 2000. Invasion of a multitude of genetic niches by mobile endonuclease genes. FEMS Microbiol Lett 185: 99–107. 10.1111/j.1574-6968.2000.tb09046.x

Gorbalenya AE, Koonin EV. 1991. Endonuclease (R) subunits of type-I and type-III restriction-modification enzymes contain a helicase-like domain. FEBS Lett 291: 277–281. 10.1016/0014-5793(91)81301-N

Gormley NA, Hillberg AL, Halford SE. 2002. The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence. J Biol Chem 277: 4034–4041. 10.1074/jbc.M108442200

Grable J, Frederick CA, Samudzi C, Jen-Jacobson L, Lesser D, Greene P, Boyer HW, Itakura K, Rosenberg JM. 1984. Two-fold symmetry of crystalline DNA-EcoRI endonuclease recognition complexes. J Biomol Struct Dyn 1: 1149–1160. 10.1080/07391102.1984.10507509

Grabowski G, Jeltsch A, Wolfes H, Maass G, Alves J. 1995. Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI. Gene 157: 113–118. 10.1016/0378-1119(94)00714-4

Grasby JA, Connolly BA. 1992. Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease. Biochemistry 31: 7855–7861. 10.1021/bi00149a016

Grazulis S, Deibert M, Rimseliene R, Skirgaila R, Sasnauskas G, Lagunavicius A, Repin V, Urbanke C, Huber R, Siksnys V. 2002. Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence. Nucleic Acids Res 30: 876–885. 10.1093/nar/30.4.876

Grigorescu A, Horvath M, Wilkosz PA, Chandrasekhar K, Rosenberg JM. 2004. The integration of recognition and cleavage: x-ray structures of pre-transition state complex, post-reactive complex, and the DNA-free endonuclease. In Restriction endonucleases (ed. Pingoud A), pp. 137–177. Springer, Berlin, Heidelberg.

Hager PW, Reich NO, Day JP, Coche TG, Boyer HW, Rosenberg JM, Greene PJ. 1990. Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements. J Biol Chem 265: 21520–21526.

Halford SE. 2001. Hopping, jumping and looping by restriction enzymes. Biochem Soc Trans 29: 363–374. 10.1042/bst0290363

Halford SE, Marko JF. 2004. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res 32: 3040–3052. 10.1093/nar/gkh624

Halford SE, Bilcock DT, Stanford NP, Williams SA, Milsom SE, Gormley NA, Watson MA, Bath AJ, Embleton ML, Gowers DM, et al. 1999. Restriction endonuclease reactions requiring two recognition sites. Biochem Soc Trans 27: 696–699. 10.1042/bst0270696

Hall MC, Matson SW. 1999. Helicase motifs: the engine that powers DNA unwinding. Mol Microbiol 34: 867–877. 10.1046/j.1365-2958.1999.01659.x

Hashimoto H, Horton JR, Zhang X, Bostick M, Jacobsen SE, Cheng X. 2008. The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix. Nature 455: 826–829. 10.1038/nature07280

Hattman S. 1973. Plasmid-controlled variation in the content of methylated bases in single-stranded DNA bacteriophages M13 and fd. J Mol Biol 74: 749–752. 10.1016/0022-2836(73)90064-8

Hedgpeth J, Goodman HM, Boyer HW. 1972. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci 69: 3448–3452. 10.1073/pnas.69.11.3448

Heitman J. 1993. On the origins, structures and functions of restriction-modification enzymes. Genet Eng 15: 57–108. 10.1007/978-1-4899-1666-2_4

Heitman J, Model P. 1990. Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition. EMBO J 9: 3369–3378. 10.1002/j.1460-2075.1990.tb07538.x

Hickman AB, Li Y, Mathew SV, May EW, Craig NL, Dyda F. 2000. Unexpected structural diversity in DNA recombination: the restriction endonuclease connection. Mol Cell 5: 1025–1034. 10.1016/S1097-2765(00)80267-1

Horton NC, Perona JJ. 1998. Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT)+d(ATCTT). J Mol Biol 277: 779–787. 10.1006/jmbi.1998.1655

Horton NC, Perona JJ. 2000. Crystallographic snapshots along a protein-induced DNA-bending pathway. Proc Natl Acad Sci 97: 5729–5734. 10.1073/pnas.090370797

Horton JR, Nastri HG, Riggs PD, Cheng X. 1998a. Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis. J Mol Biol 284: 1491–1504. 10.1006/jmbi.1998.2269

Horton NC, Newberry KJ, Perona JJ. 1998b. Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. Proc Natl Acad Sci 95: 13489–13494. 10.1073/pnas.95.23.13489

Horton NC, Dorner LF, Perona JJ. 2002. Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. Nat Struct Biol 9: 42–47. 10.1038/nsb741

Horton JR, Blumenthal RM, Cheng X. 2004a. Restriction endonucleases: structure of the conserved catalytic core and the role of metal ions in DNA cleavage. In Restriction endonucleases (ed. Pingoud A), pp. 361–392. Springer, Berlin.

Horton JR, Ratner G, Banavali NK, Huang N, Choi Y, Maier MA, Marquez VE, MacKerell AD Jr, Cheng X. 2004b. Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase. Nucleic Acids Res 32: 3877–3886. 10.1093/nar/gkh701

Horton JR, Zhang X, Maunus R, Yang Z, Wilson GG, Roberts RJ, Cheng X. 2006. DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion. Nucleic Acids Res 34: 939–948. 10.1093/nar/gkj484

Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X. 2014. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix. Nucleic Acids Res 42: 12092–12101. 10.1093/nar/gku871

Hsieh PC, Xiao JP, O'Loane D, Xu SY. 2000. Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI. J Bacteriol 182: 949–955. 10.1128/JB.182.4.949-955.2000

Huai Q, Colandene JD, Chen Y, Luo F, Zhao Y, Topal MD, Ke H. 2000. Crystal structure of NaeI—an evolutionary bridge between DNA endonuclease and topoisomerase. EMBO J 19: 3110–3118. 10.1093/emboj/19.12.3110

Huai Q, Colandene JD, Topal MD, Ke H. 2001. Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement. Nat Struct Biol 8: 665–669. 10.1038/90366

Humbelin M, Suri B, Rao DN, Hornby DP, Eberle H, Pripfl T, Kenel S, Bickle TA. 1988. Type III DNA restriction and modification systems EcoP1 and EcoP15. Nucleotide sequence of the EcoP1 operon, the EcoP15 mod gene and some EcoP1 mod mutants. J Mol Biol 200: 23–29. 10.1016/0022-2836(88)90330-0

Hurst GD, Werren JH. 2001. The role of selfish genetic elements in eukaryotic evolution. Nat Rev Genet 2: 597–606. 10.1038/35084545

Ivanenko T, Heitman J, Kiss A. 1998. Mutational analysis of the function of Met137 and Ile197, two amino acids implicated in sequence-specific DNA recognition by the EcoRI endonuclease. Biol Chem 379: 459–465. 10.1515/bchm.1998.379.4-5.459

Iwasaki H, Han YW, Okamoto T, Ohnishi T, Yoshikawa M, Yamada K, Toh H, Daiyasu H, Ogura T, Shinagawa H. 2000. Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for holliday junction branch migration. Mol Microbiol 36: 528–538. 10.1046/j.1365-2958.2000.01842.x

Janscak P, Dryden DT, Firman K. 1998. Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I. Nucleic Acids Res 26: 4439–4445. 10.1093/nar/26.19.4439

Janscak P, MacWilliams MP, Sandmeier U, Nagaraja V, Bickle TA. 1999a. DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes. EMBO J 18: 2638–2647. 10.1093/emboj/18.9.2638

Janscak P, Sandmeier U, Bickle TA. 1999b. Single amino acid substitutions in the HsdR subunit of the type IB restriction enzyme EcoAI uncouple the DNA translocation and DNA cleavage activities of the enzyme. Nucleic Acids Res 27: 2638–2643. 10.1093/nar/27.13.2638

Janscak P, Sandmeier U, Szczelkun MD, Bickle TA. 2001. Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I. J Mol Biol 306: 417–431. 10.1006/jmbi.2000.4411

Janulaitis A, Stakenas P, Berlin Yu A. 1983. A new site-specific endodeoxyribonuclease from Citrobacter freundii. FEBS Lett 161: 210–212. 10.1016/0014-5793(83)81009-6

Janulaitis A, Vaisvila R, Timinskas A, Klimasauskas S, Butkus V. 1992. Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes. Nucleic Acids Res 20: 6051–6056. 10.1093/nar/20.22.6051

Jeltsch A. 1999. Circular permutations in the molecular evolution of DNA methyltransferases. J Mol Evol 49: 161–164. 10.1007/PL00006529

Jeltsch A, Pingoud A. 1996. Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems. J Mol Evol 42: 91–96. 10.1007/BF02198833

Jeltsch A, Urbanke C. 2004. Sliding or hopping? How restriction enzymes find their way on DNA. In Restriction endonucleases (ed. Pingoud A), pp. 95–110. Springer, Berlin.

Jeltsch A, Alves J, Maass G, Pingoud A. 1992. On the catalytic mechanism of EcoRI and EcoRV. A detailed proposal based on biochemical results, structural data and molecular modelling. FEBS Lett 304: 4–8. 10.1016/0014-5793(92)80576-3

Jeltsch A, Alves J, Oelgeschläger T, Wolfes H, Maass G, Pingoud A. 1993a. Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis. J Mol Biol 229: 221–234. 10.1006/jmbi.1993.1019

Jeltsch A, Alves J, Wolfes H, Maass G, Pingoud A. 1993b. Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc Natl Acad Sci 90: 8499–8503. 10.1073/pnas.90.18.8499

Jeltsch A, Kroger M, Pingoud A. 1995a. Evidence for an evolutionary relationship among type-II restriction endonucleases. Gene 160: 7–16. 10.1016/0378-1119(95)00181-5

Jeltsch A, Maschke H, Selent U, Wenz C, Köhler E, Connolly BA, Thorogood H, Pingoud A. 1995b. DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme. Biochemistry 34: 6239–6246. 10.1021/bi00018a028

Jen-Jacobson L. 1997. Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44: 153–180. 10.1002/(SICI)1097-0282(1997)44:2<153::AID-BIP4>3.0.CO;2-U

Jen-Jacobson L, Engler LE, Lesser DR, Kurpiewski MR, Yee C, McVerry B. 1996. Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites. EMBO J 15: 2870–2882. 10.1002/j.1460-2075.1996.tb00648.x

Jen-Jacobson L, Lesser D.R., Kurpiewski M. 1991. DNA sequence discrimination by EcoRI endonuclease. In Nucleic acids and molecular biology (ed. Eckstein F, Lilley DMJ), pp. 142–170. Springer, Heidelberg.

Jen-Jacobson L, Engler LE, Jacobson LA. 2000. Structural and thermodynamic strategies for site-specific DNA binding proteins. Structure 8: 1015–1023. 10.1016/S0969-2126(00)00501-3

Jindrova E, Schmid-Nuoffer S, Hamburger F, Janscak P, Bickle TA. 2005. On the DNA cleavage mechanism of Type I restriction enzymes. Nucl Acids Res 33: 1760–1766. 10.1093/nar/gki322

Jo K, Topal MD. 1995. DNA topoisomerase and recombinase activities in Nae I restriction endonuclease. Science 267: 1817–1820. 10.1126/science.7892605

Johnson MS, Sutcliffe MJ, Blundell TL. 1990. Molecular anatomy: phyletic relationships derived from three-dimensional structures of proteins. J Mol Evol 30: 43–59. 10.1007/BF02102452

Jones S, Daley DT, Luscombe NM, Berman HM, Thornton JM. 2001. Protein–RNA interactions: a structural analysis. Nucleic Acids Res 29: 943–954. 10.1093/nar/29.4.943

Jose TJ, Conlan LH, Dupureur CM. 1999. Quantitative evaluation of metal ion binding to PvuII restriction endonuclease. J Biol Inorg Chem 4: 814–823. 10.1007/s007750050355

Kandavelou K, Chandrasegaran S. 2009. Custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes. Methods Mol Biol 544: 617–636. 10.1007/978-1-59745-483-4_40

Kandavelou K, Mani M, Durai S, Chandrasegaran S. 2005. “Magic” scissors for genome surgery. Nat Biotechnol 23: 686–687. 10.1038/nbt0605-686

Kandavelou K, Mani M, Durai S, Chandrasegaran S. 2004. Engineering and applications of chemeric nucleases. In Restriction endonucleases (ed. Pingoud A), pp. 413–434. Springer, Berlin.

Kandavelou K, Ramalingam S, London V, Mani M, Wu J, Alexeev V, Civin CI, Chandrasegaran S. 2009. Targeted manipulation of mammalian genomes using designed zinc finger nucleases. Biochem Biophys Res Commun 388: 56–61. 10.1016/j.bbrc.2009.07.112

Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM. 1990. Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. Science 249: 1307–1309. 10.1126/science.2399465

Kim Y, Choi J, Grable JC, Greene P, Hager P, Rosenberg JM. 1994. In Structural biology: the state of the art (ed. Sarma RH, Sarma MH), pp. 225–246. Adenine Press, Schenectady, NY.

Kim DE, Narayan M, Patel SS. 2002. T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J Mol Biol 321: 807–819. 10.1016/S0022-2836(02)00733-7

King G, Murray NE. 1994. Restriction enzymes in cells, not eppendorfs. Trends Microbiol 2: 465–469. 10.1016/0966-842X(94)90649-1

King K, Benkovic SJ, Modrich P. 1989. Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease. J Biol Chem 264: 11807–11815.

Kirsanova OV, Baskunov VB, Gromova ES. 2004. Type IIE and IIF restriction endonucleases interacting with two recognition sites on DNA. Mol Biol 38: 886–900. 10.1023/B:MBIL.0000043944.45429.aa

Kita K, Kotani H, Sugisaki H, Takanami M. 1989. The FokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes. J Biol Chem 264: 5751–5756.

Klimasauskas S, Kumar S, Roberts RJ, Cheng X. 1994. HhaI methyltransferase flips its target base out of the DNA helix. Cell 76: 357–369. 10.1016/0092-8674(94)90342-5

Kobayashi I. 1996. DNA modification and restriction: selfish behavior of an epigenetic system. In Epigenetic mechanisms of gene regulation (ed. Russo V, Martienssen R, Riggs A), pp. 155–172. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Kobayashi I. 1998. Selfishness and death: raison d'etre of restriction, recombination and mitochondria. Trends Genet 14: 368–374. 10.1016/S0168-9525(98)01532-7

Kobayashi I. 2001. Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res 29: 3742–3756. 10.1093/nar/29.18.3742

Kobayashi I. 2004. Restriction-modification systems as minimal forms of life. In Restriction endonucleases (ed. Pingoud A), pp. 19–62. Springer, Berlin.

Kong H. 1998. Analyzing the functional organization of a novel restriction modification system, the BcgI system. J Mol Biol 279: 823–832. 10.1006/jmbi.1998.1821

Kong H, Smith CL. 1997. Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, BcgI. Nucleic Acids Res 25: 3687–3692. 10.1093/nar/25.18.3687

Korolev S, Hsieh J, Gauss GH, Lohman TM, Waksman G. 1997. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90: 635–647. 10.1016/S0092-8674(00)80525-5

Kostrewa D, Winkler FK. 1995. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 Å resolution. Biochemistry 34: 683–696. 10.1021/bi00002a036

Kosykh VG, Puntezhis SA, Bur'ianov Ia I, Baev AA. 1982. [Isolation, purification and properties of restriction endonuclease EcoRII]. Biokhimiia (Moscow, Russia) 47: 619–625.

Kovall RA, Matthews BW. 1998. Structural, functional, and evolutionary relationships between λ-exonuclease and the type II restriction endonucleases. Proc Natl Acad Sci 95: 7893–7897. 10.1073/pnas.95.14.7893

Kovall RA, Matthews BW. 1999. Type II restriction endonucleases: structural, functional and evolutionary relationships. Curr Opin Chem Biol 3: 578–583. 10.1016/S1367-5931(99)00012-5

Krüger DH, Bickle TA. 1983. Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts. Microbiol Rev 47: 345–360.

Krüger DH, Barcak GJ, Reuter M, Smith HO. 1988. EcoRII can be activated to cleave refractory DNA recognition sites. Nucleic Acids Res 16: 3997–4008. 10.1093/nar/16.9.3997

Krüger DH, Schroeder C, Reuter M, Bogdarina IG, Buryanov YI, Bickle TA. 1985. DNA methylation of bacterial viruses T3 and T7 by different DNA methylases in Escherichia coli K12 cells. EUr J Biochem/FEBS 150: 323–330. 10.1111/j.1432-1033.1985.tb09024.x

Krüger DH, Kupper D, Meisel A, Reuter M, Schroeder C. 1995. The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes. FEMS Microbiol Rev 17: 177–184. 10.1111/j.1574-6976.1995.tb00200.x

Kupper D, Reuter M, Mackeldanz P, Meisel A, Alves J, Schroeder C, Krüger DH. 1995. Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites. Protein Expression Purif 6: 1–9. 10.1006/prep.1995.1001

Kuster W. 1998. “Bedeutung hydrophober kontakte fur die sequenzspezifische DNA-erkennung der restriktionsendonuklease EcoRI.” Doctoral thesis, University of Hanover.

Kuz'min NP, Loseva SP, Beliaeva R, Kravets AN, Solonin AS. 1984. [EcoRV restrictase: physical and catalytic properties of homogenous enzyme]. Mol Biol (Mosc) 18: 197–204.

Lacks S, Greenberg B. 1975. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J Biol Chem 250: 4060–4066.

Lesser DR, Kurpiewski MR, Jen-Jacobson L. 1990. The energetic basis of specificity in the EcoRI endonuclease–DNA interaction. Science 250: 776–786. 10.1126/science.2237428

Lesser DR, Kurpiewski MR, Waters T, Connolly BA, Jen-Jacobson L. 1993. Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition. Proc Natl Acad Sci 90: 7548–7552. 10.1073/pnas.90.16.7548

Loenen WA, Dryden DT, Raleigh EA, Wilson GG. 2014a. Type I restriction enzymes and their relatives. Nucleic Acids Res 42: 20–44. 10.1093/nar/gkt847

Loenen WA, Dryden DT, Raleigh EA, Wilson GG, Murray NE. 2014b. Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res 42: 3–19. 10.1093/nar/gkt990

Loenen WAM. 2003. Tracking EcoKI and DNA fifty years on: a golden story full of surprises. Nucleic Acids Res 31: 7059–7069. 10.1093/nar/gkg944

Looney MC, Moran LS, Jack WE, Feehery GR, Benner JS, Slatko BE, Wilson GG. 1989. Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase. Gene 80: 193–208. 10.1016/0378-1119(89)90284-9

Lukacs CM, Aggarwal AK. 2001. BglII and MunI: what a difference a base makes. Curr Opin Struct Biol 11: 14–18. 10.1016/S0959-440X(00)00174-3

Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK. 2000. Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 Å resolution. Nat Struct Biol 7: 134–140. 10.1038/72405

Luscombe NM, Laskowski RA, Thornton JM. 2001. Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Res 29: 2860–2874. 10.1093/nar/29.13.2860

Mahdi AA, Briggs GS, Sharples GJ, Wen Q, Lloyd RG. 2003. A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins. EMBO J 22: 724–734. 10.1093/emboj/cdg043

Makovets S, Titheradge AJ, Murray NE. 1998. ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems. Mol Microbiol 28: 25–35. 10.1046/j.1365-2958.1998.00767.x

Makovets S, Doronina VA, Murray NE. 1999. Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes. Proc Natl Acad Sci 96: 9757–9762. 10.1073/pnas.96.17.9757

Maluf NK, Fischer CJ, Lohman TM. 2003. A dimer of Escherichia coli UvrD is the active form of the helicase in vitro. J Mol Biol 325: 913–935. 10.1016/S0022-2836(02)01277-9

Mani M, Kandavelou K, Dy FJ, Durai S, Chandrasegaran S. 2005a. Design, engineering, and characterization of zinc finger nucleases. Biochem Biophys Res Commun 335: 447–457. 10.1016/j.bbrc.2005.07.089

Mani M, Smith J, Kandavelou K, Berg JM, Chandrasegaran S. 2005b. Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem Biophys Res Commun 334: 1191–1197. 10.1016/j.bbrc.2005.07.021

Martin AM, Sam MD, Reich NO, Perona JJ. 1999. Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease. Nat Struct Biol 6: 269–277. 10.1038/8195

McClelland SE, Szczelkun MD. 2004. The Type I and III restriction endonucleases: structural elements in molecular motors that process DNA. In Restriction endonucleases (ed. Pingoud A), pp. 111–135. Springer, Berlin.

McKane M, Milkman R. 1995. Transduction, restriction and recombination patterns in Escherichia coli. Genetics 139: 35–43.

Meisel A, Mackeldanz P, Bickle TA, Krüger DH, Schroeder C. 1995. Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis. EMBO J 14: 2958–2966. 10.1002/j.1460-2075.1995.tb07296.x

Mernagh DR, Kneale GG. 1996. High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site. Nucleic Acids Res 24: 4853–4858. 10.1093/nar/24.24.4853

Mernagh DR, Taylor IA, Kneale GG. 1998. Interaction of the type I methyltransferase M.EcoR124I with modified DNA substrates: sequence discrimination and base flipping. Biochem J 336 (Pt 3): 719–725. 10.1042/bj3360719

Milsom SE, Halford SE, Embleton ML, Szczelkun MD. 2001. Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites. J Mol Biol 311: 515–527. 10.1006/jmbi.2001.4893

Mizuuchi K, Nobbs TJ, Halford SE, Adzuma K, Qin J. 1999. A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase. Biochemistry 38: 4640–4648. 10.1021/bi990054p

Mordasini T, Curioni A, Andreoni W. 2003. Why do divalent metal ions either promote or inhibit enzymatic reactions? The case of BamHI restriction endonuclease from combined quantum-classical simulations. J Biol Chem 278: 4381–4384. 10.1074/jbc.C200664200

Mucke M, Lurz R, Mackeldanz P, Behlke J, Krüger DH, Reuter M. 2000. Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage. J Biol Chem 275: 30631–30637. 10.1074/jbc.M003904200

Mucke M, Grelle G, Behlke J, Kraft R, Krüger DH, Reuter M. 2002. EcoRII: a restriction enzyme evolving recombination functions? EMBO J 21: 5262–5268. 10.1093/emboj/cdf514

Mucke M, Krüger DH, Reuter M. 2003. Diversity of type II restriction endonucleases that require two DNA recognition sites. Nucleic Acids Res 31: 6079–6084. 10.1093/nar/gkg836

Muir RS, Flores H, Zinder ND, Model P, Soberon X, Heitman J. 1997. Temperature-sensitive mutants of the EcoRI endonuclease. J Mol Biol 274: 722–737. 10.1006/jmbi.1997.1419

Murray NE. 2000. Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64: 412–434. 10.1128/MMBR.64.2.412-434.2000

Murray NE. 2002. 2001 Fred Griffith review lecture. Immigration control of DNA in bacteria: self versus non-self. Microbiology 148: 3–20. 10.1099/00221287-148-1-3

Murray NE, Daniel AS, Cowan GM, Sharp PM. 1993. Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes. Mol Microbiol 9: 133–143. 10.1111/j.1365-2958.1993.tb01675.x

Naito T, Kusano K, Kobayashi I. 1995. Selfish behavior of restriction-modification systems. Science 267: 897–899. 10.1126/science.7846533

Nakayama Y, Kobayashi I. 1998. Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion. Proc Natl Acad Sci 95: 6442–6447. 10.1073/pnas.95.11.6442

Nanduri B, Byrd AK, Eoff RL, Tackett AJ, Raney KD. 2002. Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor. Proc Natl Acad Sci 99: 14722–14727. 10.1073/pnas.232401899

Needels MC, Fried SR, Love R, Rosenberg JM, Boyer HW, Greene PJ. 1989. Determinants of EcoRI endonuclease sequence discrimination. Proc Natl Acad Sci. 86: 3579–3583. 10.1073/pnas.86.10.3579

Newman PC, Nwosu VU, Williams DM, Cosstick R, Seela F, Connolly BA. 1990a. Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase. Biochemistry 29: 9891–9901. 10.1021/bi00494a020

Newman PC, Williams DM, Cosstick R, Seela F, Connolly BA. 1990b. Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). Biochemistry 29: 9902–9910. 10.1021/bi00494a021

Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK. 1994a. Structure of restriction endonuclease BamHI and its relationship to EcoRI. Nature 368: 660–664. 10.1038/368660a0

Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK. 1994b. Structure of restriction endonuclease BamHI phased at 1.95 A resolution by MAD analysis. Structure 2: 439–452. 10.1016/S0969-2126(00)00045-9

Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK. 1995. Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269: 656–663. 10.1126/science.7624794

Newman M, Lunnen K, Wilson G, Greci J, Schildkraut I, Phillips SE. 1998. Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence. EMBO J 17: 5466–5476. 10.1093/emboj/17.18.5466

Nobbs TJ, Halford SE. 1995. DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distant DNA sites. J Mol Biol 252: 399–411. 10.1006/jmbi.1995.0506

Nobusato A, Uchiyama I, Kobayashi I. 2000a. Diversity of restriction-modification gene homologues in Helicobacter pylori. Gene 259: 89–98. 10.1016/S0378-1119(00)00455-8

Nobusato A, Uchiyama I, Ohashi S, Kobayashi I. 2000b. Insertion with long target duplication: a mechanism for gene mobility suggested from comparison of two related bacterial genomes. Gene 259: 99–108. 10.1016/S0378-1119(00)00456-X

Nunes-Duby SE, Kwon HJ, Tirumalai RS, Ellenberger T, Landy A. 1998. Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26: 391–406. 10.1093/nar/26.2.391

O'Neill M, Chen A, Murray NE. 1997. The restriction-modification genes of Escherichia coli K-12 may not be selfish: they do not resist loss and are readily replaced by alleles conferring different specificities. Proc Natl Acad Sci 94: 14596–14601. 10.1073/pnas.94.26.14596

O'Neill M, Dryden DT, Murray NE. 1998. Localization of a protein–DNA interface by random mutagenesis. EMBO J 17: 7118–7127. 10.1093/emboj/17.23.7118

O'Neill M, Powell LM, Murray NE. 2001. Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity. J Mol Biol 307: 951–963. 10.1006/jmbi.2001.4543

Oelgeschläger T, Geiger R, Rüter T, Alves J, Fliess A, Pingoud A. 1990. Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants. Gene 89: 19–27. 10.1016/0378-1119(90)90201-2

Oller AR, Vanden Broek W, Conrad M, Topal MD. 1991. Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochemistry 30: 2543–2549. 10.1021/bi00223a035

Pabo CO, Peisach E, Grant RA. 2001. Design and selection of novel Cys2His2 zinc finger proteins. Ann Rev Biochem 70: 313–340. 10.1146/annurev.biochem.70.1.313

Panne D, Raleigh EA, Bickle TA. 1999. The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis. J Mol Biol 290: 49–60. 10.1006/jmbi.1999.2894

Park JS, Marr MT, Roberts JW. 2002. E. coli transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation. Cell 109: 757–767. 10.1016/S0092-8674(02)00769-9

Pein CD, Reuter M, Cech D, Krüger DH. 1989. Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII. FEBS Lett 245: 141–144. 10.1016/0014-5793(89)80208-X

Pein CD, Reuter M, Meisel A, Cech D, Krüger DH. 1991. Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA. Nucleic Acids Res 19: 5139–5142. 10.1093/nar/19.19.5139

Petrauskene OV, Karpova EA, Gromova ES, Guschlbauer W. 1994. Two subunits of EcoRII restriction endonuclease interact with two DNA recognition sites. Biochem Biophys Res Commun 198: 885–890. 10.1006/bbrc.1994.1126

Petrauskene OV, Babkina OV, Tashlitsky VN, Kazankov GM, Gromova ES. 1998. EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event. FEBS Lett 425: 29–34. 10.1016/S0014-5793(98)00184-7

Piatrauskene OV, Tashlitskii VN, Brevnov MG, Bakman I, Gromova ES. 1996. [Kinetic modeling of the mechanism of allosteric interactions of restriction endonuclease EcoRII with two DNA segments]. Biokhimiia (Moscow, Russia) 61: 1257–1269.

Piekarowicz A, Golaszewska M, Sunday AO, Siwinska M, Stein DC. 1999. The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a single polypeptide. J Mol Biol 293: 1055–1065. 10.1006/jmbi.1999.3198

Pieper U, Pingoud A. 2002. A mutational analysis of the PD…D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli. Biochemistry 41: 5236–5244. 10.1021/bi0156862

Pieper U, Schweitzer T, Groll DH, Pingoud A. 1999. Defining the location and function of domains of McrB by deletion mutagenesis. Biol Chem 380: 1225–1230. 10.1515/BC.1999.155

Pinarbasi H, Pinarbasi E, Hornby DP. 2003. The small subunit of M · AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain. J Bacteriol 185: 1284–1288. 10.1128/JB.185.4.1284-1288.2003

Pingoud A. 2004. Restriction endonucleases. Springer, Berlin.

Pingoud A, Jeltsch A. 1997. Recognition and cleavage of DNA by type-II restriction endonucleases. Eur J Biochem/FEBS 246: 1–22. 10.1111/j.1432-1033.1997.t01-6-00001.x

Pingoud A, Jeltsch A. 2001. Structure and function of type II restriction endonucleases. Nucleic Acids Res 29: 3705–3727. 10.1093/nar/29.18.3705

Pingoud V, Kubareva E, Stengel G, Friedhoff P, Bujnicki JM, Urbanke C, Sudina A, Pingoud A. 2002. Evolutionary relationship between different subgroups of restriction endonucleases. J Biol Chem 277: 14306–14314. 10.1074/jbc.M111625200

Pingoud A, Fuxreiter M, Pingoud V, Wende W. 2005. Type II restriction endonucleases: structure and mechanism. Cell Mol Life Sci 62: 685–707. 10.1007/s00018-004-4513-1

Ponting CP, Russell RR. 2002. The natural history of protein domains. Ann Rev Biophys Biomol Struct 31: 45–71. 10.1146/annurev.biophys.31.082901.134314

Porteus MH, Baltimore D. 2003. Chimeric nucleases stimulate gene targeting in human cells. Science 300: 763. 10.1126/science.1078395

Powell LM, Murray NE. 1995. S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase. Nucleic Acids Res 23: 967–974. 10.1093/nar/23.6.967

Powell LM, Dryden DT, Willcock DF, Pain RH, Murray NE. 1993. DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine. J Mol Biol 234: 60–71. 10.1006/jmbi.1993.1563

Powell LM, Connolly BA, Dryden DT. 1998a. The DNA binding characteristics of the trimeric EcoKI methyltransferase and its partially assembled dimeric form determined by fluorescence polarisation and DNA footprinting. J Mol Biol 283: 947–961. 10.1006/jmbi.1998.2142

Powell LM, Dryden DT, Murray NE. 1998b. Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme. J Mol Biol 283: 963–976. 10.1006/jmbi.1998.2143

Powell LM, Lejeune E, Hussain FS, Cronshaw AD, Kelly SM, Price NC, Dryden DT. 2003. Assembly of EcoKI DNA methyltransferase requires the C-terminal region of the HsdM modification subunit. Biophys Chem 103: 129–137. 10.1016/S0301-4622(02)00251-X

Prakash-Cheng A, Ryu J. 1993. Delayed expression of in vivo restriction activity following conjugal transfer of Escherichia coli hsdK (restriction-modification) genes. J Bacteriol 175: 4905–4906. 10.1128/jb.175.15.4905-4906.1993

Prakash-Cheng A, Chung SS, Ryu J. 1993. The expression and regulation of hsdK genes after conjugative transfer. Mol Gen Genet 241: 491–496. 10.1007/BF00279890

Price C, Bickle TA. 1986. A possible role for DNA restriction in bacterial evolution. Microbiol Sci 3: 296–299.

Putnam CD, Clancy SB, Tsuruta H, Gonzalez S, Wetmur JG, Tainer JA. 2001. Structure and mechanism of the RuvB Holliday junction branch migration motor. J Mol Biol 311: 297–310. 10.1006/jmbi.2001.4852

Raleigh EA, Wilson G. 1986. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci 83: 9070–9074. 10.1073/pnas.83.23.9070

Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PW, Elhai J, Hanahan D. 1988. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res 16: 1563–1575. 10.1093/nar/16.4.1563

Ramalingam S, Kandavelou K, Rajenderan R, Chandrasegaran S. 2011. Creating designed zinc-finger nucleases with minimal cytotoxicity. J Mol Biol 405: 630–641. 10.1016/j.jmb.2010.10.043

Rao DN, Saha S, Krishnamurthy V. 2000. ATP-dependent restriction enzymes. Prog Nucleic Acid Res Mol Biol 64: 1–63. 10.1016/S0079-6603(00)64001-1

Reid SL, Parry D, Liu HH, Connolly BA. 2001. Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization. Biochemistry 40: 2484–2494. 10.1021/bi001956p

Repin VE, Lebedev LR, Puchkova L, Serov GD, Tereschenko T, Chizikov VE, Andreeva I. 1995. New restriction endonucleases from thermophilic soil bacteria. Gene 157: 321–322. 10.1016/0378-1119(94)00781-M

Reuter M, Kupper D, Meisel A, Schroeder C, Krüger DH. 1998. Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites. J Biol Chem 273: 8294–8300. 10.1074/jbc.273.14.8294

Reuter M, Schneider-Mergener J, Kupper D, Meisel A, Mackeldanz P, Krüger DH, Schroeder C. 1999. Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires. J Biol Chem 274: 5213–5221. 10.1074/jbc.274.8.5213

Reuter M, Mucke M, Krüger DH. 2004. Structure and function of Type IIE restriction endonucleases—or: from a plasmid that restricts phage replication to a new molecular DNA recognition mechanism. In Restriction endonucleases (ed. Pingoud A), pp. 261–295. Springer, Berlin.

Rigden DJ, Setlow P, Setlow B, Bagyan I, Stein RA, Jedrzejas MJ. 2002. PrfA protein of Bacillus species: prediction and demonstration of endonuclease activity on DNA. Protein Sci 11: 2370–2381. 10.1110/ps.0216802

Rippe K, von Hippel PH, Langowski J. 1995. Action at a distance: DNA-looping and initiation of transcription. Trends Biochem Sci 20: 500–506. 10.1016/S0968-0004(00)89117-3

Roberts RJ, Cheng X. 1998. Base flipping. Ann Rev Bochem 67: 181–198. 10.1146/annurev.biochem.67.1.181

Roberts RJ, Halford S.E. 1993. Type II restriction enzymes. In Nucleases, 2nd ed. (ed. Linn SM, Lloyd RS, Roberts RJ), pp. 35–88. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Roberts RJ, Macelis D. 1991. Restriction enzymes and their isoschizomers. Nucleic Acids Res 19 (Suppl): 2077–2109. 10.1093/nar/19.suppl.2077

Roberts RJ, Macelis D. 1993. REBASE—restriction enzymes and methylases. Nucleic Acids Res 21: 3125–3137. 10.1093/nar/21.13.3125

Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev S, Dryden DT, Dybvig K, et al. 2003a. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res 31: 1805–1812. 10.1093/nar/gkg274

Roberts RJ, Vincze T, Posfai J, Macelis D. 2003b. REBASE: restriction enzymes and methyltransferases. Nucleic Acids Res 31: 418–420. 10.1093/nar/gkg069

Roberts RJ, Macelis D, Vincze T, Pósfai J. 2004. The genomics of restriction and modification. In 5th NEB Meeting on Restriction and Modification (Bristol UK, 2004). http://rebase.neb.com/cgi-bin/statlist.

Roman LJ, Kowalczykowski SC. 1989. Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA. Biochemistry 28: 2873–2881. 10.1021/bi00433a019

Rosati O. 1999. “Untersuchung und design von DNA-kontakten der restriktionsendonuklease EcoRI inner- und ausserhalb der erkennungssequenz.” Doctoral thesis, University of Hanover.

Rosenberg JM. 1991. Structure and function of restriction endonucleases. Curr Opin Struct Biol 1: 104–113. 10.1016/0959-440X(91)90018-O

Saha S, Rao DN. 1995. ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme. J Mol Biol 247: 559–567.

Saha S, Rao DN.1997. Mutations in the Res subunit of the EcoPI restriction enzyme that affect ATP-dependent reactions. J Mol Biol 269: 342–354. 10.1006/jmbi.1997.1045

Saha S, Ahmad I, Reddy YV, Krishnamurthy V, Rao DN. 1998. Functional analysis of conserved motifs in type III restriction-modification enzymes. Biol Chem 379: 511–517. 10.1515/bchm.1998.379.4-5.511

Samudzi CT. 1990. “Use of the molecular replacement method in structural studies of EcoRI endonuclease.” PhD thesis, University of Pittsburgh.

Sapranauskas R, Sasnauskas G, Lagunavicius A, Vilkaitis G, Lubys A, Siksnys V. 2000. Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium. J Biol Chem 275: 30878–30885. 10.1074/jbc.M003350200

Scheuring Vanamee E, Viadiu H, Lukacs CM, Aggarwal AK. 2004. Two of a kind: BamHI and BglII. In Restriction endonucleases (ed. Pingoud A), pp. 215–236. Springer, Berlin.

Schildkraut I, Banner CD, Rhodes CS, Parekh S. 1984. The cleavage site for the restriction endonuclease EcoRV is 5′-GAT/ATC-3′. Gene 27: 327–329. 10.1016/0378-1119(84)90078-7

Schleif R. 1992. DNA looping. Ann Rev Biochem 61: 199–223. 10.1146/annurev.bi.61.070192.001215

Schulze C, Jeltsch A, Franke I, Urbanke C, Pingoud A. 1998. Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover. EMBO J 17: 6757–6766. 10.1093/emboj/17.22.6757

Selent U, Rüter T, Köhler E, Liedtke M, Thielking V, Alves J, Oelgeschläger T, Wolfes H, Peters F, Pingoud A. 1992. A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV. Biochemistry 31: 4808–4815. 10.1021/bi00135a010

Senesac JH, Allen JR. 1995. Oligonucleotide activation of the type IIe restriction enzyme NaeI for digestion of refractory sites. BioTechniques 19: 990–993.

Sharp PM, Kelleher JE, Daniel AS, Cowan GM, Murray NE. 1992. Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria. Proc Natl Acad Sci 89: 9836–9840. 10.1073/pnas.89.20.9836

Sidorova NY, Rau DC. 1996. Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences. Proc Natl Acad Sci 93: 12272–12277. 10.1073/pnas.93.22.12272

Sidorova N, Rau D.C. 2004. The role of water in the EcoRI-DNA binding. In Restriction endonucleases (ed. Pingoud A), pp. 319–337. Springer, Berlin.

Šikšnys V, Zareckaja N, Vaisvila R, Timinskas A, Stakenas P, Butkus V, Janulaitis A. 1994. CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R·MunI and R·EcoRI. Gene 142: 1–8. 10.1016/0378-1119(94)90347-6

Šikšnys V, Timinskas A, Klimasauskas S, Butkus V, Janulaitis A. 1995. Sequence similarity among type-II restriction endonucleases, related by their recognized 6-bp target and tetranucleotide-overhang cleavage. Gene 157: 311–314. 10.1016/0378-1119(94)00632-3

Šikšnys V, Skirgaila R, Sasnauskas G, Urbanke C, Cherny D, Grazulis S, Huber R. 1999. The Cfr10I restriction enzyme is functional as a tetramer. J Mol Biol 291: 1105–1118. 10.1006/jmbi.1999.2977

Šikšnys V, Grazulis S., Huber R. 2004. Structure and function of the tetrameric restriction enzymes. In Restriction endonucleases (ed. Pingoud A), pp. 237–259. Springer, Berlin.

Singleton MR, Wigley DB. 2002. Modularity and specialization in superfamily 1 and 2 helicases. J Bacteriol 184: 1819–1826. 10.1128/JB.184.7.1819-1826.2002

Singleton MR, Wigley DB. 2003. Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes. EMBO J 22: 4579–4583. 10.1093/emboj/cdg441

Singleton MR, Scaife S, Wigley DB. 2001. Structural analysis of DNA replication fork reversal by RecG. Cell 107: 79–89. 10.1016/S0092-8674(01)00501-3

Skirgaila R, Grazulis S, Bozic D, Huber R, Siksnys V. 1998. Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues. J Mol Biol 279: 473–481. 10.1006/jmbi.1998.1803

Stahl F, Wende W, Jeltsch A, Pingoud A. 1996. Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein. Proc Natl Acad Sci 93: 6175–6180. 10.1073/pnas.93.12.6175

Stahl F, Wende W, Jeltsch A, Pingoud A. 1998a. The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis. Biol Chem 379: 467–473. 10.1515/bchm.1998.379.4-5.467

Stahl F, Wende W, Wenz C, Jeltsch A, Pingoud A. 1998b. Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit. Biochemistry 37: 5682–5688. 10.1021/bi973025s

Stanford NP, Halford SE, Baldwin GS. 1999. DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis. J Mol Biol 288: 105–116. 10.1006/jmbi.1999.2673

Stankevicius K, Lubys A, Timinskas A, Vaitkevicius D, Janulaitis A. 1998. Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes. Nucleic Acids Res 26: 1084–1091. 10.1093/nar/26.4.1084

Stasiak A. 1980a. [Restriction enzymes. I. Mechanisms of action of type II restriction-modification systems (author's transl)]. Postępy Biochem 26: 343–367.

Stasiak A. 1980b. [Restriction enzymes. II. Mechanisms of action of type I and III restriction-modification systems (author's transl)]. Postępy Biochem 26: 369–387.

Stein DC, Chien R, Seifert HS. 1992. Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification. J Bacteriol 174: 4899–4906. 10.1128/jb.174.15.4899-4906.1992

Stewart FJ, Raleigh EA. 1998. Dependence of McrBC cleavage on distance between recognition elements. Biol Chem 379: 611–616.

Stewart FJ, Panne D, Bickle TA, Raleigh EA. 2000. Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme. J Mol Biol 298: 611–622. 10.1006/jmbi.2000.3697

Stover T, Köhler E, Fagin U, Wende W, Wolfes H, Pingoud A. 1993. Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+. J Biol Chem 268: 8645–8650.

Strzelecka T, Newman M, Dorner LF, Knott R, Schildkraut I, Aggarwal AK. 1994. Crystallization and preliminary X-ray analysis of restriction endonuclease BamHI-DNA complex. J Mol Biol 239: 430–432. 10.1006/jmbi.1994.1383

Stuckey JA, Dixon JE. 1999. Crystal structure of a phospholipase D family member. Nat Struct Biol 6: 278–284. 10.1038/6716

Studier FW, Bandyopadhyay PK. 1988. Model for how type I restriction enzymes select cleavage sites in DNA. Proc Natl Acad Sci 85: 4677–4681. 10.1073/pnas.85.13.4677

Sturrock SS, Dryden DT. 1997. A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes. Nucleic Acids Res 25: 3408–3414. 10.1093/nar/25.17.3408

Su TJ, Tock MR, Egelhaaf SU, Poon WC, Dryden DT. 2005. DNA bending by M·EcoKI methyltransferase is coupled to nucleotide flipping. Nucleic Acids Res 33: 3235–3244. 10.1093/nar/gki618

Subramanya HS, Bird LE, Brannigan JA, Wigley DB. 1996. Crystal structure of a DExx box DNA helicase. Nature 384: 379–383. 10.1038/384379a0

Sugisaki H, Kanazawa S. 1981. New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI). Gene 16: 73–78. 10.1016/0378-1119(81)90062-7

Sun J, Viadiu H, Aggarwal AK, Weinstein H. 2003. Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex. Biophys J 84: 3317–3325. 10.1016/S0006-3495(03)70056-3

Szczelkun MD. 2000. How do proteins move along DNA? Lessons from type-I and type-III restriction endonucleases. Essays in biochemistry: motor proteins (ed. Banting G, Higgins SJ), Vol. 35, pp. 131–143. Portland Press, London.

Szczelkun MD, Connolly BA. 1995. Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues. Biochemistry 34: 10724–10733. 10.1021/bi00034a004

Szczelkun MD, Halford SE. 1996. Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease. EMBO J 15: 1460–1469. 10.1002/j.1460-2075.1996.tb00488.x

Szczelkun MD, Dillingham MS, Janscak P, Firman K, Halford SE. 1996. Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates. EMBO J 15: 6335–6347. 10.1002/j.1460-2075.1996.tb01023.x

Szybalski W, Kim SC, Hasan N, Podhajska AJ. 1991. Class-IIS restriction enzymes—a review. Gene 100: 13–26. 10.1016/0378-1119(91)90345-C

Takano T, Watanabe T, Fukasawa T. 1968. Mechanism of host-controlled restriction of bacteriophage λ by R factors in Escherichia coli K12. Virology 34: 290–302. 10.1016/0042-6822(68)90239-0

Tamulaitis G, Solonin AS, Siksnys V. 2002. Alternative arrangements of catalytic residues at the active sites of restriction enzymes. FEBS Lett 518: 17–22. 10.1016/S0014-5793(02)02621-2

Tanner NK, Cordin O, Banroques J, Doere M, Linder P. 2003. The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol Cell 11: 127–138. 10.1016/S1097-2765(03)00006-6

Taylor JD, Halford SE. 1989. Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry 28: 6198–6207. 10.1021/bi00441a011

Taylor JD, Badcoe IG, Clarke AR, Halford SE. 1991. EcoRV restriction endonuclease binds all DNA sequences with equal affinity. Biochemistry 30: 8743–8753. 10.1021/bi00100a005

Theis K, Chen PJ, Skorvaga M, Van Houten B, Kisker C. 1999. Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair. EMBO J 18: 6899–6907. 10.1093/emboj/18.24.6899

Thielking V, Alves J, Fliess A, Maass G, Pingoud A. 1990. Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry 29: 4682–4691. 10.1021/bi00471a024

Thielking V, Selent U, Köhler E, Wolfes H, Pieper U, Geiger R, Urbanke C, Winkler FK, Pingoud A. 1991. Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. Biochemistry 30: 6416–6422. 10.1021/bi00240a011

Thielking V, Selent U, Köhler E, Landgraf A, Wolfes H, Alves J, Pingoud A. 1992. Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease. Biochemistry 31: 3727–3732. 10.1021/bi00130a001

Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS. 1999. Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group. Nucleic Acids Res 27: 3438–3445. 10.1093/nar/27.17.3438

Thorogood H, Grasby JA, Connolly BA. 1996. Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates. J Biol Chem 271: 8855–8862. 10.1074/jbc.271.15.8855

Titheradge AJ, Ternent D, Murray NE. 1996. A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA. Mol Microbiol 22: 437–447. 10.1046/j.1365-2958.1996.00126.x

Titheradge AJ, King J, Ryu J, Murray NE. 2001. Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems. Nucleic Acids Res 29: 4195–4205. 10.1093/nar/29.20.4195

Todd AE, Orengo CA, Thornton JM. 2002. Plasticity of enzyme active sites. Trends Biochem Sci 27: 419–426. 10.1016/S0968-0004(02)02158-8

Topal MD, Conrad M. 1993. Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases. Nucleic Acids Res 21: 2599–2603. 10.1093/nar/21.11.2599

Topal MD, Thresher RJ, Conrad M, Griffith J. 1991. NaeI endonuclease binding to pBR322 DNA induces looping. Biochemistry 30: 2006–2010. 10.1021/bi00221a038

Tsutakawa SE, Jingami H, Morikawa K. 1999a. Recognition of a TG mismatch: the crystal structure of very short patch repair endonuclease in complex with a DNA duplex. Cell 99: 615–623. 10.1016/S0092-8674(00)81550-0

Tsutakawa SE, Muto T, Kawate T, Jingami H, Kunishima N, Ariyoshi M, Kohda D, Nakagawa M, Morikawa K. 1999b. Crystallographic and functional studies of very short patch repair endonuclease. Mol Cell 3: 621–628. 10.1016/S1097-2765(00)80355-X

van der Woerd MJ, Pelletier JJ, Xu S, Friedman AM. 2001. Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis. Structure 9: 133–144. 10.1016/S0969-2126(01)00564-0

Van Heuverswyn H, Fiers W. 1980. Recognition sequence for the restriction endonuclease BglI from Bacillus globigii. Gene 9: 195–203. 10.1016/0378-1119(90)90322-I

Vanamee ES, Viadiu H, Kucera R, Dorner L, Picone S, Schildkraut I, Aggarwal AK. 2005. A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA. EMBO J 24: 4198–4208. 10.1038/sj.emboj.7600880

Velankar SS, Soultanas P, Dillingham MS, Subramanya HS, Wigley DB. 1999. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97: 75–84. 10.1016/S0092-8674(00)80716-3

Venclovas C, Timinskas A, Siksnys V. 1994. Five-stranded β-sheet sandwiched with two α-helices: a structural link between restriction endonucleases EcoRI and EcoRV. Proteins 20: 279–282. 10.1002/prot.340200308

Vermote CL, Halford SE. 1992. EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition. Biochemistry 31: 6082–6089. 10.1021/bi00141a018

Viadiu H, Aggarwal AK. 1998. The role of metals in catalysis by the restriction endonuclease BamHI. Nat Struct Biol 5: 910–916. 10.1038/2352

Viadiu H, Aggarwal AK. 2000. Structure of BamHI bound to nonspecific DNA: a model for DNA sliding. Mol Cell 5: 889–895. 10.1016/S1097-2765(00)80329-9

Viadiu H, Vanamee ES, Jacobson EM, Schildkraut I, Aggarwal AK. 2003. Crystallization of restriction endonuclease SfiI in complex with DNA. Acta Crystallogr, Sect D: Biol Crystallogr 59: 1493–1495. 10.1107/S0907444903011910

Vilkaitis G, Lubys A, Merkiene E, Timinskas A, Janulaitis A, Klimasauskas S. 2002. Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation. Nucleic Acids Res 30: 1547–1557. 10.1093/nar/30.7.1547

Vinogradova MV, Gromova ES, Kosykh VG, Bur'ianov Ia I, Shabarova ZA. 1990. [Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. Determination of the size of EcoRII binding site]. Molekuliarnaia Biologiia 24: 847–850.

Vipond IB, Halford SE. 1993. Structure–function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage. Molr Microbiol 9: 225–231. 10.1111/j.1365-2958.1993.tb01685.x

Vipond IB, Halford SE. 1995. Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions. Biochemistry 34: 1113–1119. 10.1021/bi00004a002

Vipond IB, Baldwin GS, Halford SE. 1995. Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases. Biochemistry 34: 697–704. 10.1021/bi00002a037

Vitkute J, Maneliene Z, Petrusyte M, Janulaitis A. 1997. BplI, a new BcgI-like restriction endonuclease, which recognizes a symmetric sequence. Nucleic Acids Res 25: 4444–4446. 10.1093/nar/25.22.4444

Vovis GF, Horiuchi K, Zinder ND. 1975. Endonuclease R-EcoRII restriction of bacteriophage f1 DNA in vitro: ordering of genes V and VII, location of an RNA promotor for gene VIII. J Virol 16: 674–684.

Wah DA, Hirsch JA, Dorner LF, Schildkraut I, Aggarwal AK. 1997. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388: 97–100. 10.1038/40446

Wah DA, Bitinaite J, Schildkraut I, Aggarwal AK. 1998. Structure of FokI has implications for DNA cleavage. Proc Natl Scad Sci 95: 10564–10569. 10.1073/pnas.95.18.10564

Walker JE, Saraste M, Runswick MJ, Gay NJ. 1982. Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1: 945–951. 10.1002/j.1460-2075.1982.tb01276.x

Wang J, Chen R, Julin DA. 2000. A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions. J Biol Chem 275: 507–513. 10.1074/jbc.275.1.507

Waters TR, Connolly BA. 1994. Interaction of the restriction endonuclease EcoRV with the deoxyguanosine and deoxycytidine bases in its recognition sequence. Biochemistry 33: 1812–1819. 10.1021/bi00173a026

Webb JL, King G, Ternent D, Titheradge AJ, Murray NE. 1996. Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs. EMBO J 15: 2003–2009. 10.1002/j.1460-2075.1996.tb00551.x

Welsh AJ, Halford SE, Scott DJ. 2004. Analysis of Type II restriction endonucleases that interact with two recognition sites. In Restriction endonucleases (ed. Pingoud A), pp. 297–317. Springer, Berlin.

Wentzell LM, Halford SE. 1998. DNA looping by the SfiI restriction endonuclease. J Mol Biol 281: 433–444. 10.1006/jmbi.1998.1967

Wentzell LM, Nobbs TJ, Halford SE. 1995. The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence. J Mol Biol 248: 581–595. 10.1006/jmbi.1995.0244

Wenz C, Jeltsch A, Pingoud A. 1996. Probing the indirect readout of the restriction enzyme EcoRV. Mutational analysis of contacts to the DNA backbone. J Biol Chem 271: 5565–5573. 10.1074/jbc.271.10.5565

West SC. 1996. DNA helicases: new breeds of translocating motors and molecular pumps. Cell 86: 177–180. 10.1016/S0092-8674(00)80088-4

Whitehouse I, Stockdale C, Flaus A, Szczelkun MD, Owen-Hughes T. 2003. Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme. Mol Cell Biol 23: 1935–1945. 10.1128/MCB.23.6.1935-1945.2003

Wilkins BM. 2000. Plasmid promiscuity: meeting the challenge of DNA immigration. Env Microbiol 4: 495–500. 10.1046/j.1462-2920.2002.00332.x

Wilkosz PA, Chandrasekhar WK, Rosenberg JM. 1995. Preliminary characterization of EcoRI-DNA co-crystals: incomplete factorial design of oligonucleotide sequences. Acta Crystallogr, Sect D: Biol Crystallogr 51: 938–945. 10.1107/S0907444994005251

Willcock DF, Dryden DT, Murray NE. 1994. A mutational analysis of the two motifs common to adenine methyltransferases. EMBO J 13: 3902–3908. 10.1002/j.1460-2075.1994.tb06701.x

Wilson GG. 1991. Organization of restriction-modification systems. Nucleic Acids Res 19: 2539–2566. 10.1093/nar/19.10.2539

Windolph S, Alves J. 1997. Influence of divalent cations on inner-arm mutants of restriction endonuclease EcoRI. Eur J Biochem/FEBS 244: 134–139. 10.1111/j.1432-1033.1997.00134.x

Winkler FK. 1992. Structure and function of restriction endonucleases. Curr Opin Struct Biol 2: 93–99. 10.1016/0959-440X(92)90183-8

Winkler FK. 1994. DNA totally flipped-out by methylase. Structure 2: 79–83. 10.1016/S0969-2126(00)00009-5

Winkler FK, Prota AE. 2004. Structure and function of EcoRV endonuclease. In Restriction endonucleases (ed. Pingoud A), pp. 179–214. Springer, Berlin.

Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS. 1993. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J 12: 1781–1795. 10.1002/j.1460-2075.1993.tb05826.x

Wolfes H, Alves J, Fliess A, Geiger R, Pingoud A. 1986. Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI. Nucleic Acids Res 14: 9063–9080. 10.1093/nar/14.22.9063

Wright DJ, King K, Modrich P. 1989. The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease. J Biol Chem 264: 11816–11821.

Wu J, Kandavelou K, Chandrasegaran S. 2007. Custom-designed zinc finger nucleases: what is next? Cell Mol Life Sci 64: 2933–2944. 10.1007/s00018-007-7206-8

Yang CC, Topal MD. 1992. Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site. Biochemistry 31: 9657–9664. 10.1021/bi00155a019

Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, Weber PC. 1997. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol 4: 463–467. 10.1038/nsb0697-463

Yolov AA, Gromova ES, Kubareva EA, Potapov VK, Shabarova ZA. 1985. Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages. Nucleic Acids Res 13: 8969–8981. 10.1093/nar/13.24.8969

Yoshimori R, Roulland-Dussoix D, Boyer HW. 1972. R factor-controlled restriction and modification of deoxyribonucleic acid: restriction mutants. J Bacteriol 112: 1275–1279.

Zaremba M, Urbanke C, Halford SE, Siksnys V. 2004. Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily. J Mol Biol 336: 81–92. 10.1016/j.jmb.2003.12.012

Zaremba M, Sasnauskas G, Urbanke C, Siksnys V. 2005. Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations. J Mol Biol 348: 459–478. 10.1016/j.jmb.2005.02.037

Zaremba M, Sasnauskas G, Urbanke C, Siksnys V. 2006. Allosteric communication network in the tetrameric restriction endonuclease Bse634I. J Mol Biol 363: 800–812. 10.1016/j.jmb.2006.08.050

Zaremba M, Sasnauskas G, Siksnys V. 2012. The link between restriction endonuclease fidelity and oligomeric state: a study with Bse634I. FEBS Lett 586: 3324–3329. 10.1016/j.febslet.2012.07.009

Zhou XE, Wang Y, Reuter M, Mucke M, Krüger DH, Meehan EJ, Chen L. 2004. Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. J Mol Biol 335: 307–319. 10.1016/j.jmb.2003.10.030


http://rebase.neb.com/rebase/rebase.html The Restriction Enzyme Database.

Get the full book:
PDF / ePub / mobi

Buy the Print Book