Restriction Enzymes: A History
By Wil A.M. Loenen, Leiden University Medical Center
April 2019 · 346 pages, illustrated (38 color and 26 B&W)
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|
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).
TYPE II ENZYMES
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
Cleaves both sides of target on both strands
Symmetric or asymmetric target. R and M functions in one polypeptide
(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
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
Symmetric or asymmetric target. R genes are heterodimers
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).
TYPE I AND III ENZYMES
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.
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http://rebase.neb.com/rebase/rebase.html The Restriction Enzyme Database.