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J. Biol. Chem., Vol. 277, Issue 6, 4024-4033, February 8, 2002

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Many Type IIs Restriction Endonucleases Interact with Two Recognition Sites before Cleaving DNA^*

Abigail J. Bath, Susan E. Milsom, Niall A. Gormley, and Stephen E. Halford

From the Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom

Received for publication, August 31, 2001, and in revised form, November 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the one-site DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Over 3000 type II restriction endonucleases have been identified to date (1). These enzymes recognize short DNA sequences, 4-8 bp long and cleave both strands of the DNA at fixed locations in or near their recognition sites (2). With one exception, BfiI (3), they require Mg²⁺ or a similar divalent metal ion to cleave DNA (4), although a few also require AdoMet¹ for maximal activity (5). Many of the type II enzymes are homodimeric proteins that interact symmetrically with palindromic DNA sequences, so that one active site in the dimer is placed to cleave one strand of the DNA and the other active site the equivalent phosphodiester bond in the opposite strand: for example, EcoRV, BamHI, and BglI (6-8). Enzymes of this sort cleave DNA with multiple target sites by means of separate reactions at each site (9), although they can act processively and migrate from one site to another by intramolecular processes (10).

Several type II enzymes that recognize palindromic sequences differ from the orthodox enzymes, because they have to interact with two copies of their target sequence before they can cleave DNA (11, 12). The latter include the type IIe enzymes, such as EcoRII, NaeI, and Sau3AI (13-17), and the type IIf enzymes, such as SfiI, SgrAI, Cfr10I, and NgoMIV (18-21). Both the type IIe and IIf enzymes bind two sites concurrently but the former cleave only one site per turnover, whereas the latter cleave both sites concertedly within a single turnover (22). Except for Sau3AI, a monomer in free solution (17), the type IIe enzymes are dimers with two distinct DNA-binding clefts, both of which bind the cognate DNA sequence but one is an allosteric locus that activates DNA cleavage in the other cleft (15, 16). In contrast, the type IIf enzymes are generally tetramers with two identical surfaces for binding their palindromic sites, each made from two subunits (18, 21).

The type II family contains another subgroup, the type IIs systems (24). The type IIs endonucleases recognize asymmetric DNA sequences, 4-7 bp long, and cleave both strands at specific locations up to 20 bases away from their recognition site (1). Several hundred type IIs enzymes have been identified, and those characterized to date are monomers in solution (25-28). Hence, they cannot act in the same way as the dimeric and tetrameric enzymes noted above.

The archetypal type IIs enzyme, FokI, consists of a DNA-binding domain and a catalytic domain, which are connected by a flexible linker (29). In the crystal structure of FokI bound to DNA, the DNA-binding domain from a single monomer covers the entire recognition sequence, but the catalytic domain lies distant from the DNA and interacts instead with the binding domain (30). The catalytic domain has the function of cleaving only one phosphodiester bond, thus posing a question: How does FokI cut both strands? However, when crystallized in the absence of DNA, the structure showed two monomers of FokI interacting with each other via their catalytic domains, with the two active sites organized like those in the BamHI dimer (31). The dimerization is necessary for DNA cleavage. The rates of cleavage of a DNA with one FokI site do not increase proportionally to increases in the enzyme concentration but instead increase more steeply, which suggests that more than one molecule of the enzyme is needed for each cleavage event (32). In addition, the catalytic domain of FokI cannot cleave DNA by itself, but it can enhance the activity of wild-type FokI, probably by associating with the catalytic domain of the latter and cleaving the second DNA strand (32). In the presence of Ca²⁺ as a non-catalytic analogue of Mg²⁺, the binding of FokI to DNA duplexes carrying the recognition sequence yields a complex containing two protomers of FokI and two duplexes (33). Nevertheless, the pathway for the assembly of a FokI dimer at its recognition site(s) has yet to be established. The monomer bound to the recognition sequence (30) could recruit a second monomer from solution or from elsewhere on either the same DNA molecule, in cis, or from another DNA molecule, in trans (31, 32). DNA cleavage by FokI may thus require a bridging interaction between two copies of its recognition sequence (33). If so, it should be more active on a DNA with two sites than on a DNA with one site, because interactions spanning two DNA sites occur more readily in cis than in trans (34).

Evidence also exists to suggest that a number of other type IIs enzymes require two sites for their cleavage reactions. For example, BspMI has a very low activity on a plasmid that has one BspMI site, but its cleavage of this plasmid is stimulated by the addition of DNA molecules that have multiple BspMI sites (14). Similarly, Eco57I can be stimulated to cleave refractory sites by the addition of an oligonucleotide duplex that carries its recognition sequence (35). In these cases, the activating DNA may provide in trans a second site for the enzyme, which then allows the enzyme to cleave the refractory site, as noted with several type IIe and type IIf nucleases (13-22).

The aim of this work was to investigate whether interactions with two DNA sites are a general feature of the reactions of the type IIs endonucleases, by testing several type IIs enzymes against plasmids with either one or two copies of the relevant recognition sequence. The orthodox type II enzymes cleave substrates with one or two sites at the same rate, and the two sites in the latter are cleaved sequentially, giving rise first to the singly cut DNA and then the doubly cut products: If the enzyme has the same activity at each site on the two-site DNA, the singly cut DNA accumulates to a maximum of 40% of the total DNA before declining to the doubly cut product (19). On the other hand, enzymes that require two copies of their recognition site will usually cleave the two-site DNA faster than the one-site DNA (12). This strategy also distinguishes the type IIe enzymes, which require two sites but cleave only one of them, from the type IIf enzymes, which act concertedly at two sites (22). The accompanying paper (36) describes further studies on the mode of action of one type IIs enzyme, BspMI, which interacts with two sites but by a different mechanism from the others described here.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins-- FokI, purified to homogeneity, was a gift from J. Bitinaite and I. Schildkraut (New England BioLabs, Beverly, MA). BspMI was purified as in the accompanying paper (36). The molar concentrations of FokI and BspMI are given in terms of the monomeric and tetrameric forms of the proteins, respectively (25, 36). Eco57I was purchased from MBI Fermentas (Vilnius, Lithuania) and Acc36I from SibEnzyme (Novosibirsk, Russia). All other enzymes were from New England BioLabs. Concentrations of the enzymes from commercial sources are given in terms of units of enzyme activity, as defined by the supplier.

DNA-- Plasmids pBR322 (37), pAT153 (38), pNEB193 (New England BioLabs), and pSKFok1 (from J. Bitinaite (32)) were manipulated by standard procedures (39) to yield the plasmids shown in Fig. 1. The constructions of these plasmids generally involved the cleavage of one of the vectors noted above with two restriction enzymes; the isolation of the requisite section of the vector by electrophoresis through agarose; and the ligation of this fragment to a duplex made by annealing two complimentary oligodeoxyribonucleotides (the duplexes had 5' single-strand extensions of four bases that matched the termini from the digest of the vector). The ligation mixtures were used to transform Escherichia coli HB101 (39), and the transformants containing the desired construct were identified by restriction analyses. The validity of the plasmids was confirmed by DNA sequencing across the site of the insertion of the oligoduplex (University of Bristol Sequencing Service). The transformants carrying the requisite plasmid were cultured in M9 minimal media with 37 MBq/liter [methyl-³H]thymidine, and the covalently closed form of the plasmid was purified by density-gradient centrifugations (18, 19). The preparations were largely the supercoiled form of the monomeric plasmid with generally <10% dimer or nicked open-circle DNA.

The oligonucleotides used in the above manipulations were pur- chased from MWG Biotech Ltd. (Milton Keynes, UK) and had the following sequences: for the conversion of pBR322 (a plasmid with single sites for BsmBI, BsmI, SapI, BsaI, and BsgI) to pML2 (a plasmid with two sites for each of these enzymes (Fig. 1), 5'-AATTCGGAGACGGTGAATGCGCTCTTCCGCGAGACCCACCCTGCACCA-3' in the top strand (consecutive recognition sites for BsmI and SapI are underlined) and 5'-AGCTTGGTGCAGGGTGGGTCTCGCGGAAGAGCGCATTCACCGTCTCCG-3' in the bottom (from left to right, recognition sites for BsgI, BsaI, and BsmBI are underlined); for the conversion of pNEB193 (one BpmI site) to pAB5 (two BpmI sites), 5'-AATTGAGACCCACGCTCACCGGCTCCAGATT-3' in the top strand and 5'-CGCGAATCTGGAGCCGGTGAGCGTGGGTCTC-3' in the bottom (BpmI site underlined); for the conversion of pSKFok1 (one FokI site) to pSKFok2 (two FokI sites), 5'-GATCCCAAGGGGGATGTGCTGCAAGGCGATT-3' in the top strand (FokI site underlined) and 5'-CTAGAATCGCCTTGCAGCACATCCCCCTTGG-3' in the bottom; to convert pAT153 (one BspMI site) to pNAG1 (two BspMI sites), 5'-AGCTGGCCATGCTGTCCAGGCAGGTAGATGAC-3' in the top strand and 5'-GATCGTCATCTACCTGCCTGGACAGCATGGCC-3' in the bottom (BspMI site underlined). Whether the recognition sequence is located in the top or the bottom strand of the above duplexes determines the orientation of that site in the plasmid. In all of the plasmids constructed here, the two sites for each enzyme were in directly repeated (head-to-tail) orientation.

A further plasmid, pSKFok3, was constructed by subjecting pSKFok2 to a partial digest with PvuII and a complete digest with HincII, prior to re-circularization by blunt-end ligation. This procedure removed one of the two FokI sites in pSKFok2, the site originally present in pSKFok1, and leaves a single FokI site, that introduced during the construction of pSKFok2 (Fig. 1).

DNA Cleavage Reactions-- Restriction enzymes were assayed on ³H-labeled DNA (5-10 nM) in 200-µl reactions, in the buffer and at the temperature advised by the supplier. The following reaction buffers were used: buffer A, 20 mM Tris acetate (pH 7.9), 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT, and 100 µg/ml BSA; buffer A+, as A with 80 µM AdoMet; buffer B, 50 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 100 µg/ml BSA; buffer C, as buffer B except for NaCl at 100 mM; buffer D, 20 mM HEPES (pH 8.0), 80 mM NaCl, 10 mM MgCl₂, and 1 mM DTT. FokI was diluted before use in 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 µg/ml BSA, and 50% (v/v) glycerol. BspMI was diluted before use in 20 mM HEPES (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 µg/ml BSA, 1 mM spermine, 0.2% (v/v) Triton X-100, and 20% (v/v) glycerol. Enzymes from commercial sources were diluted in the buffers advised by the supplier. A 15-µl aliquot of the reaction mixture was removed before adding the enzyme and further aliquots taken at various times after adding the enzyme. The samples were mixed immediately with 10 µl of stop mix (9) prior to electrophoresis through agarose under conditions that separated the supercoiled substrate and each of the reaction products. The segments of agarose that contained each DNA species were analyzed by scintillation counting to evaluate the concentration of each form of DNA at each time point (22).

DNA Methylation-- Following a procedure described previously (5), 2 units of either BsgI or BpmI were added to four parallel samples containing 2 µg of phage  DNA in 20 mM Tris acetate (pH 7.9), 50 mM potassium acetate, 1 mM DTT, and 100 mg/ml BSA. One sample contained no further components: the second was supplemented with 80 µM AdoMet; the third with 10 mM magnesium acetate; and the fourth with both 80 µM AdoMet and 10 mM magnesium acetate. The samples were left for 1 h at 37 °C, prior to heating to 67 °C for 20 min and cooling on ice. To the samples that lacked Mg²⁺, magnesium acetate was added to a final concentration of 10 mM along with a second aliquot of the same enzyme, and these were incubated for a further period of 1 h at 37 °C. The samples were analyzed by electrophoresis through agarose containing ethidium bromide, and the extent of cleavage was assessed visually under UV illumination.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The objective of this study was to characterize the steady-state reactions of several type IIs restriction endonucleases on plasmids that have either one or two copies of the relevant recognition sites, to identify whether the enzyme in question needs to interact with two recognition sites before cleaving DNA. The type IIs endonucleases examined here, and their recognition sequences, are listed in Table I. Plasmids with one copy of each of these recognition sequences were already available (Table I), and these were used to construct new plasmids with two copies of the requisite sequence (Fig. 1 and Table I). In some instances (Fig. 1a), the sequences around each site in the two-site plasmid were identical for 2 or 3 bp immediately adjacent to the recognition sequence (Table I). However, restriction endonucleases can be influenced by the sequences flanking the recognition site (2), and the type IIs enzymes may also be affected by the sequence throughout the length of DNA between recognition and cleavage sites. Consequently, wherever practicable (Fig. 1, b-d), the two recognition sites had the same flanking sequences for 3 bp upstream of the recognition site and for all of the downstream sequence to 2 bp beyond the sites of cleavage (Table I).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Plasmid substrates. a, pBR322, which contains one site for BsmBI, BsmI, SapI, BsaI, and BsgI at the positions indicated, was converted to pML2 by inserting between the EcoRI and HindIII sites an oligonucleotide duplex with a second copy of each site. b, pNEB193, which contains one site for BpmI, was converted to pAB5 by inserting between the EcoRI and AscI sites a duplex containing a BpmI site. c, pSKFok1 has one recognition site for FokI and was converted to a plasmid with two sites, pSKFok2, by inserting a duplex between the BamHI and XbaI sites; pSKFok3 was created from pSKFok2 by using PvuII and HincII to delete the segment of DNA carrying the FokI site originally present in pSKFok1, so as to leave only the FokI site inserted during the construction of pSKFok2. d, pAT153, which contains a single BspMI site, was converted to pNAG1 by inserting between the HindIII and BamHI sites a duplex carrying a BspMI site.

In the plasmids with two sites for a given enzyme, the length of DNA between the sites varied from 189 to 2134 bp (Table I), but this will not be a significant factor in determining whether the enzyme can interact with two sites. In supercoiled DNA, the juxtaposition of two sites in three-dimensional space is largely independent of the length of the DNA between the sites (40, 41). Because the recognition sequences for type IIs enzymes are asymmetric, a further factor that might affect the ability of a type IIs enzyme to interact with two sites is the relative orientation of the sites, because several systems that act at two DNA sites require sites in a specified orientation (42). Preliminary experiments in this laboratory have revealed that both FokI and BspMI cleave substrates with two sites in direct repeat at different rates from substrates with two sites in inverted orientation (data not shown). Hence, to permit comparisons between the enzymes studied here, the two-site substrates all had sites in directly repeated orientation.

The FokI and BspMI enzymes were purified to homogeneity and were studied under steady-state conditions, with the enzyme at a known concentration below that of the substrate. Only a small fraction of the DNA would then be bound to the enzyme at any time during the reaction, and the majority of the DNA products observed in the course of the reaction are free species liberated from the enzyme. The other enzymes were purchased from commercial sources and were supplied at concentrations specified in terms of units of enzyme activity: The molar concentrations of these proteins were not known. However, whenever tested, the reaction velocities of these enzymes increased as the number of units of enzyme added to the reaction increased. Hence, it is likely that their reactions were also carried out under steady-state conditions with the enzymes at lower concentrations than the DNA.

DNA Cleavage by BsmBI, BsmI, BsaI, and SapI-- These enzymes were selected as examples of type IIs nucleases that cleave DNA close to their recognition sites, 1 bp away in one strand and 5 bp away in the other (Table I). The target sites for these four enzymes occur once in pBR322, and a plasmid with two copies of each recognition sequence was constructed from pBR322 (Fig. 1a). The enzymes were tested against ³H-labeled preparations of the two plasmids. Samples were taken from the reactions at timed intervals and analyzed as under "Experimental Procedures" to determine the concentrations of the supercoiled substrate and all of the various reaction products (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   BsmBI on plasmids with one or two recognition sites. The drawings indicate the various forms of DNA that can exist during the reaction of a restriction enzyme on circular DNA with either one (a) or two (b) recognition sites: SC, supercoiled substrate; OC, open-circle DNA cleaved in one strand; FLL, full-length linear DNA cleaved in both strands at one site; L1 and L2 (only in b), the two linear DNA products from cutting both strands at both sites. The reactions contained 48 units/ml BsmBI and 5 nM DNA (~90% supercoiled) in buffer C at 55 °C. The DNA was: in a, pBR322 (which has one BsmBI site); in b, pML2 (which has two BsmBI sites). At timed intervals after adding the enzyme, aliquots were removed from the reactions and analyzed as under "Experimental Procedures" to obtain concentrations of the following forms of DNA: , supercoiled substrate (marked SC on both graphs); , open-circle DNA (marked OC); , full-length linear DNA (marked FLL); , total DNA cut at both sites (marked in b as L1/2).

BsmBI cleaved the supercoiled (SC) form of pBR322 directly to the full-length linear (FLL) form, without the accumulation of any of the nicked open-circle (OC) DNA during the course of the reaction (Fig. 2a). Hence, BsmBI cuts its recognition site in both strands within a single DNA binding event. The plasmid with two BsmBI sites was cleaved first at one site, to give the FLL form, again without liberating any of the OC DNA; after a lag phase, the remaining site was then cleaved in a separate reaction to yield the two final products, L1 and L2 (Fig. 2b). The time course for the formation and decay of the FLL form during the reaction on the two-site substrate matches that expected for two separate reactions, with the intrinsic rate for cutting the first site equal to that for the second (9, 19). In addition, the initial rate for the consumption of the SC DNA with one BsmBI site was similar to that for the substrate with two sites (Table II).

When BsmI, BsaI, and SapI were tested against the plasmids with one or with two copies of their respective recognition sites (reactions not shown), they all behaved in essentially the same manner as that shown for BsmBI (Fig. 2). In particular, these enzymes cleaved the substrates with one and two sites at similar rates (Table II (12)), and they cut the two-site DNA by means of independent reactions at each site: In all cases, the maximal yield of FLL DNA generated during the course of their reactions on the two-site substrate was again ~40% of the total DNA, which indicates similar rates for cutting the first and second sites. If these enzymes had acted processively, first cutting one site and then translocating to the second site without leaving the DNA molecule, the maximal yield of the FLL DNA would have been <40% of the total (9, 19).

BsmBI, BsmI, BsaI, and SapI all cleave DNA in the manner of an orthodox type II restriction enzyme, such as EcoRV, BamHI, and BglI (9). They cut both strands of the DNA at an individual recognition site in one DNA binding event and that they cut two recognition sites in the same DNA sequentially, first at one site and then in a separate reaction at the second site.

DNA Cleavage by BsgI and BpmI-- In contrast to the type IIs enzymes noted above, which cleave DNA close to their recognition sites, BsgI and BpmI cut the DNA 16 and 14 bases away from their target sites, in the "top" and "bottom" strands, respectively (Table I). The recognition sequences for BsgI and BpmI are both similar to that for Eco57I, CTGAAG(16/14) (see Table I), which also cleaves DNA 16 and 14 bp away from its target site. The type IIs restriction-modification systems require two methyltransferase activities, one for each strand of their asymmetric recognition sites (43), but Eco57I differs from most type IIs systems in that a single polypeptide carries both the endonuclease and one of the methyltransferase activities (44). Both the methyltransferase and the endonuclease activities of Eco57I require AdoMet. Hence, during Eco57I reactions in the presence of AdoMet, a fraction of the DNA is protected from the endonuclease by methylation (5). BsgI and BpmI have similar genetic organizations and homologous amino acid sequences to Eco57I.² DNA cleavage by BsgI is also stimulated by AdoMet,² although BpmI has yet to be tested in this regard (1).

When the Eco57I endonuclease was tested against plasmids with one or two Eco57I sites (not shown), the principal product was the OC form of the DNA nicked in just one strand, and only a small fraction of the DNA was cleaved in both strands at either one or both sites, presumably as a consequence of the competing nuclease and methyltransferase activities of this protein. It was therefore impossible to determine whether Eco57I has the same or a higher activity on its two-site substrate compared with its one-site substrate. In contrast, both BsgI (Fig. 3) and BpmI (data not shown) made double-strand breaks at each recognition site on both their one-site and two-site substrates. In the presence of AdoMet, the initial rate of consumption of the two-site substrate for BsgI was >20 times faster than that for the DNA with one BsgI site (Table II). A possible explanation of this behavior is that the BsgI site introduced into pBR322 to create the DNA with two sites, pML2 (Fig. 1a), is more susceptible to BsgI than the site in pBR322: It has a different sequence between the recognition site and the sites of DNA cleavage 16/14 bp away. To test this possibility, pML2 was cut with another restriction enzyme to yield a linear fragment with two BsgI sites. The initial reaction of BsgI on this linear DNA yields different products depending on whether it occurs at site 1, the original site in pBR322, or at site 2, the newly introduced site (Fig. 4a). When the linear DNA was used as a substrate for BsgI, >80% of the initial cleavages occurred at the original site and <20% at the new site (Fig. 4b). The BsgI site introduced in the construction of pML2 is thus less, rather than more, susceptible than the site in pBR322. Yet the presence of this additional site in the DNA causes a major increase in the rate at which BsgI cleaves its site from pBR322.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   BsgI on plasmids with one or two sites. The reactions contained 1.2 units/ml BsgI and 5 nM DNA (~95% supercoiled) in buffer A+ at 37 °C. The DNA was: in a, pBR322 (one BsgI site); in b, pML2 (two BsgI sites). At timed intervals after adding the enzyme, aliquots were removed from the reactions and analyzed as before to obtain concentrations of the following forms of DNA: , supercoiled substrate (marked SC on both graphs); , open-circle DNA (marked OC); , full-length linear DNA (marked FLL); , total DNA cut at both sites (marked in b as L1/2).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   BsgI on linear DNA with two sites. a, the diagram shows two pathways for the reactions of BsgI on AflIII-linearized pML2: the linear DNA can be cut at site 1, the original site present in pBR322, to yield fragments A and BC; or at site 2, the additional site present in pML2, to yield fragments AB and C; cleavage at whichever site is not cut in the initial reaction then releases the three final products, A, B, and C. The number by each fragment indicates its size in bp. b, the reaction contained 4 units/ml BsgI and 5 nM linearized pML2 in buffer A+ at 37 °C. (Prior to the reaction, the SC form of pML2 had been cleaved with AflIII to yield the linear DNA with two BsgI sites at the positions indicated in a.) At timed intervals, aliquots were removed from the reaction, quenched with stop mix, and analyzed by electrophoresis through agarose to separate all of the forms of the DNA shown in a. The plot shows the concentrations of two partial products: , BC, generated by cutting only site 1; , AB, generated by cutting only site 2.

BsgI Thus Requires for Its Optimal Activity Two Copies of Its Recognition Sequence in Cis-- However, the reaction profile of BsgI on the two-site plasmid (Fig. 3b) indicates a sequential process, with the enzyme first cleaving one site and then, in a separate reaction, the remaining site. Moreover, the yield of FLL DNA during the BsgI reaction on its two-site substrate, at ~40% of the total DNA, shows that the rate for cutting the residual site is similar to that for the initial site. This profile is distinct from those for the type IIe and the type IIf enzymes that interact with two DNA sites but which, respectively, cut just one site or two sites concertedly (22).

The reaction profiles of BpmI on its one-site and two-site substrates (not shown) were like those for BsgI (Fig. 3). The rate at which BpmI consumed the two-site substrate was again much faster than that for the one-site substrate (Table II (12)). BpmI and BsgI are thus clearly similar to each other, but the enzymes were affected differently by AdoMet. The rates at which BpmI cleaved its one-site and its two-site substrates were not altered by the addition by AdoMet (data not shown). In contrast, the omission of AdoMet from BsgI reactions caused ~10-fold reductions in the rates at which it cleaved plasmids with either one or two sites (data not shown). Nevertheless, although AdoMet enhanced the rates of BsgI reactions, BsgI still cleaved its two-site substrate in the absence of AdoMet ~20 times faster than its one-site substrate.

In further studies, BsgI and BpmI were incubated with their DNA substrates in the presence of AdoMet but in the absence of Mg²⁺, to determine whether they can methylate their recognition sites, as noted previously with Eco57I (5). However, the preincubations of the DNA with AdoMet and either BsgI or BpmI failed to confer any protection of the DNA against subsequent digestions of the DNA by these enzymes in the presence of Mg²⁺ (data not shown). It thus seems that neither the BsgI nor the BpmI endonucleases are capable of methylating DNA, which presumably accounts for why their reactions, unlike those of Eco57I, proceed to completion in the presence of AdoMet.

DNA Cleavage by FokI-- The FokI endonuclease was initially tested against two plasmids: one with a single recognition site for the enzyme, pSKFok1 (32); and a second that was constructed from pSKFok1 by inserting an additional FokI site to give the two-site plasmid, pSKFok2 (Fig. 1c). FokI cleaved the two-site substrate >10 times faster than the one-site substrate (Figs. 5, a and b; Table II). However, the reaction profile for FokI on its two-site substrate (Fig. 5b) differed from that for BsgI (Fig. 3b): the amount of FLL DNA that accumulated during the course of the FokI reaction on its two-site substrate, almost 80% of the total DNA, was much larger than that with BsgI. Hence, FokI cleaves its two-site substrate initially at one site, at a rapid rate, and then at the remaining site at a much slower rate. To determine if this is due to differences in the local sequences around each site, the site originally present in pSKFok1 was deleted from pSKFok2 so as to leave a second one-site plasmid with just the newly introduced site, pSKFok3 (Fig. 1c). When the new site was the only FokI site in the plasmid, the DNA was cleaved at a slow rate (Fig. 5c), similar to that on the initial one-site plasmid (Table II). The FokI endonuclease thus has no intrinsic preference for one site in pSKFok2 over the other, yet the presence of both sites in the same DNA results in the cleavage of one of the two sites at a much faster rate than a single site in a one-site substrate. Moreover, the amount of FLL DNA produced during the reaction of FokI on pSKFok2 reached a maximum at ~8 nM, in considerable excess of the molarity of the enzyme used here, 1 nM (Fig. 5b). The FLL form therefore cannot be an enzyme-bound intermediate on the pathway to the product cut at both sites but must instead be liberated from the enzyme after cutting just one site. The reaction profile of FokI on the two-site substrate is similar to that for a type IIe enzyme, where one site serves to activate the enzyme for DNA cleavage at a second site (22).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   FokI on plasmids with one or two sites. The reactions contained 1 nM FokI and 10 nM DNA in buffer A at 37 °C. The DNA was: in a, pSKFok1 (~95% supercoiled), which has one FokI site; in b, pSKFok2 (~85% supercoiled), which has an additional FokI site; in c, pSKFok3 (~95% supercoiled), which has one FokI site, which was introduced during the construction of pSKFok2 from pSKFok1. At timed intervals after adding the enzyme, aliquots were removed from the reactions and analyzed as before to obtain concentrations of the following forms of DNA: , supercoiled substrate (marked SC on all three graphs); , open-circle DNA (marked OC); , full-length linear DNA (marked FLL); , total DNA cut at both sites (marked in b as L1/2).

The reactions of FokI on its one-site and two-site substrates were also studied across a range of enzyme concentrations, although keeping the enzyme at a lower concentration than the DNA so as to maintain steady-state conditions. To permit a comparison between the one- and the two-site plasmids, the reaction velocities were measured from the decline in the concentration of the SC substrate with time rather than from the formation of the final products, because the final products from the one- and the two-site substrates differ from each other. As shown previously (32), the velocities of the FokI reactions on a DNA with one FokI site did not increase linearly with the enzyme concentration but instead increased more steeply than expected for a linear dependence (Fig. 6a). When tested on the DNA with two FokI sites, the reaction velocities again increased with increasing enzyme concentration, by larger factors than expected for a linear dependence (Fig. 6b). However, the extents to which the increasing concentrations of FokI enhanced the reaction velocities on the one-site substrate were similar to those on the two-site substrate: The ratio of the rates on the two substrates remained constant at ~10:1 across the range of FokI concentrations.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   FokI concentration dependences. The reactions contained 10 nM DNA in buffer A at 37 °C and the concentration of FokI indicated. The DNA was: in a, pSKFok1 (one FokI site); in b, pSKFok2 (two FokI sites). For each reaction, the initial rate for substrate utilization was measured as in Fig. 5, and the values were plotted against the enzyme concentration (due to the large difference in rates on the one- and two-site substrates at each enzyme concentration tested, both x- and y-scales in a differ from those in b). The lines illustrate a linear increase in reaction rate with enzyme concentration, from the value obtained at the lowest enzyme concentration tested.

DNA Cleavage by BspMI and Acc36I-- BspMI cleaves certain plasmids with a single copy of its recognition sequence at very low rates, but it can be activated to cleave these plasmids by a second DNA with multiple BspMI sites (14). The activation should be more effective with two BspMI sites in cis, so BspMI might cleave a DNA with two recognition sites more rapidly than a DNA with one site. When tested against plasmids with either one or two BspMI sites (Fig. 1d), the plasmid with one site was indeed cleaved more slowly than that with two sites, as measured from the initial rate for the decline in the concentration of the respective substrates with time (Figs. 7, a and b). In this respect, BspMI is similar to BsgI and FokI, which also cleave their two-site substrates more rapidly than their one-site substrates (Table II). However, the amount of FLL DNA produced during the course of the BspMI reaction on its two-site substrate (Fig. 7b) was much lower than had been observed with either BsgI (Fig. 3b) or FokI (Fig. 5b). With BspMI, the initial rate for forming the FLL DNA cut at a single site was slower than that for forming the two final products cut in both strands at both sites, L1 and L2. This contrasts to the behavior of both BsgI and FokI, which convert their two-site substrates first to the DNA cut at one site and only later to the products cut at both sites. BspMI thus acts on its two-site substrate in a highly concerted manner, generally cleaving both strands at both sites within a single DNA binding event so as to convert the majority of SC plasmid directly to the final products L1 and L2. The lack of FLL DNA during the BspMI reaction on its two-site substrate could be due to processive action, but processivity cannot account for why the rate of utilization of the two-site DNA is faster than that of the one-site DNA (9, 19). Hence, BspMI must follow the same mode of action as SfiI and the other type IIf restriction enzymes (18-22).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   BspMI on plasmids with one or two recognition sites. The reactions contained 0.25 nM BspMI and 5 nM DNA (~90% supercoiled) in buffer D at 37 °C. The DNA was: in a, pAT153 (one BspMI site); in b, pNAG1 (two BspMI sites). At timed intervals after adding the enzyme, aliquots were removed from the reactions and analyzed to obtain concentrations of the following forms of DNA: , supercoiled substrate (marked SC on both graphs); , open-circle DNA (marked OC); , full-length linear DNA (marked FLL); , total DNA cut at both sites (marked in b as L1/2).

Acc36I is an isoschizomer of BspMI with respect to both its recognition sequence and its sites for DNA cleavage (Table I) and might thus be expected to act in the same way as BspMI. To see if this is so, the plasmids with one and two BspMI sites (Fig. 1d) were re-used as substrates for Acc36I. Like BspMI, Acc36I cleaved the DNA with one site more slowly than that with two sites (Table II). However, during its reaction on the two-site substrate (not shown), Acc36I liberated much more of the FLL DNA than had been observed in the BspMI reaction. The reaction profile of Acc36I on its two-site substrate was similar to that of BsgI (Fig. 3b), particularly with respect to the kinetics for the formation and decay of the FLL form of the DNA during the course of the reaction. Thus, rather than acting concertedly on DNA with two sites, Acc36I cleaves its two-site substrate in the same manner as BsgI: i.e., by means of two kinetically separate reactions that have similar rates but which are both faster than that on a DNA with a single site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The orthodox type II restriction enzymes, such as BamHI and EcoRV, are homodimeric proteins that, in most cases, interact symmetrically with their palindromic recognition sequences (2, 12). The contacts between one subunit of the protein and one half of the recognition sequence are usually, but not always, duplicated by the second subunit with the other half of the DNA, and the two active sites of the dimer are each positioned to cleave one strand of the DNA (6-8). In contrast, the type IIs restriction enzymes have asymmetric recognition sequences. It is difficult to imagine how two subunits of a homodimeric enzyme could each recognize one half of an asymmetric sequence. Instead, it is far more likely that the recognition of the complete target sequence for a type IIs enzyme is achieved by a single protein subunit. Moreover, many type IIs proteins are monomers in solution (25-28). The question then arises as to how they are able to cleave both DNA strands. It has been suggested that they may have two active sites per monomer (24), but the crystal structure of FokI shows that this is not the case, at least for this particular enzyme (30). Each FokI monomer has the catalytic functions for cutting just one strand, and it appears that FokI has to dimerize before cleaving DNA (31-33).

In this study, the kinetics of DNA cleavage by several type IIs restriction enzymes were examined under steady-state conditions, so as to reveal the form(s) of the DNA liberated from the enzyme after each turnover, as opposed to the enzyme-bound form(s). For each enzyme, two substrates were used: one plasmid with a single recognition site for the enzyme in question and another with two sites (Fig. 1; Table I). Several, but not all, of the enzymes examined cleaved the plasmid with two copies of the recognition site faster than the plasmid with one copy, which indicates that the optimal activity of these enzymes requires a bridging interaction between two DNA sites in cis (9, 19). The enzymes that cleaved their two-site substrates more rapidly than their one-site substrates showed a variety of different reaction profiles on their two-site substrates, with respect to the formation and decay of the FLL DNA cleaved at a single site. Among the nine enzymes examined, four distinct reaction schemes were observed, as judged from the rates at which they cleaved their one-site and two-site substrates and the relative rates at which they cleaved each site on the two-site plasmid. The four schemes are noted as A, B, C, and D (Table II).

Scheme A, BsmBI, BsmI, BsaI, and SapI-- One mode of action is exemplified by BsmBI, BsmI, BsaI, and SapI. All four of these enzymes cleaved their one-site substrates directly to the FLL DNA, without liberating the nicked OC form of the DNA cut in just one strand (Fig. 2a). Moreover, these enzymes cleaved each site on their two-site substrates in separate reactions (Fig. 2b), which both proceeded at the same rate as that on the one-site substrate. Unlike the other type IIs enzymes studied here, they do not need to interact with two sites. Hence, BsmBI, BsmI, BsaI, and SapI act in essentially the same manner as the orthodox type II enzymes such as EcoRV and BglI (9). The absence of OC DNA during their reactions show that they cut both strands before dissociating from the DNA. This mode of action is, however, difficult to reconcile to the general view that type IIs restriction enzymes are monomeric proteins with the catalytic functions for cutting one just strand.

One possibility is that BsmBI, BsmI, BsaI, and SapI are monomeric proteins but with two active sites per subunit, like the PI-SceI endonuclease (45). Although some of the LAGLIDADG family of homing endonucleases are homodimers with one copy of this active-site motif in each subunit, for example I-CreI (46), others such as PI-SceI are monomers with two active sites in one polypeptide, each with a copy of the LAGLIDADG motif (45). Alternatively, BsmBI, BsmI, BsaI, and SapI may be homodimeric proteins with one active site in each subunit. If so, both subunits might participate in the DNA cleavage reaction, although only one would be involved in DNA recognition. A further possibility is that these enzymes are composed of two different subunits, both of which have catalytic functions but only one has the DNA-recognition unit.³ To identify how these enzymes operate, their subunit structures and active-site organizations need to be determined, but such studies have yet to be conducted on any one of these four enzymes.

The recognition sequences for BsmBI and BsaI differ by just 1 bp whereas that for SapI is an extended version of a similar sequence (Table I). Hence, the similarities in the mode of action of BsmBI, BsaI, and SapI may be due to familial relationships among these three enzymes, like those between N.BstNBI, MlyI, and PleI (28). BsmI also cleaves DNA close to its recognition site. These four enzymes cleave one strand just 1 bp away from the recognition site. Consequently, the DNA-recognition and catalytic domains of these enzymes may not be physically separate from each other, as is the case with FokI (29, 30). The mode of action typified by BsmBI may thus be common among the type IIs enzymes that cleave DNA close to their recognition sites.

Scheme B, BsgI, BpmI, and Acc36I-- A second mode of action for type IIs enzymes was found with BsgI and BpmI and with Acc36I (Table II). These enzymes cleaved their two-site substrates more rapidly than their one-site substrates (Fig. 3), even when the site introduced to make the two-site substrate was less susceptible to the enzyme than the site originally present in the one-site DNA (Fig. 4). Hence, these enzymes require two copies of their recognition sites in cis for their optimal DNA cleavage activities. However, having cut one site in their two-site substrates, BsgI and its kin cleave the residual site, not at the rate characteristic of their reactions on a one-site DNA, but instead at a similar rate to their initial reactions on the two-site DNA: This is shown by the maximal yield of FLL DNA during their reactions on two-site substrates (Fig. 3b). One explanation for this behavior stems from the fact that the type IIs enzymes cleave the DNA away from their recognition sites, 16 and 14 bp away in the case of BsgI and BpmI. The orthodox type II enzymes usually destroy their recognition sequences by cutting the DNA within the sequence, but after a type IIs enzyme has cut one site in a two-site substrate, the DNA still possesses two intact copies of the recognition sequence. The activation of the enzyme by a second copy of its recognition site may therefore still be achievable even after the enzyme has cleaved the DNA downstream of that site. For the cleaved locus to function as an activator, the energy from the interactions with the DNA at the site of cleavage would need to be minor compared with those at the recognition site.

The BsgI and BpmI endonucleases are similar to Eco57I (5, 44) in terms of their recognition sequences (1), their genetic organizations, and their amino acid sequences² and probably also in their mode of action. Indeed, the activation of Eco57I by oligoduplexes requires the duplex to contain the recognition site, but the downstream cleavage site is not needed (35). Nevertheless, they seem to differ from each other with respect to their responses to AdoMet. Although the Eco57I protein functions as both a methyltransferase and an endonuclease, with both activities requiring AdoMet, no methylation of the DNA was observed here with either BsgI or BpmI, and, of these two, only BsgI was stimulated by AdoMet. These differences may be more apparent than real. During the purification of DNA methyltransferases, AdoMet can remain bound to the protein throughout the preparation (47). We cannot exclude the possibility that the samples of BpmI used here contain a stoichiometric amount of AdoMet, which might be sufficient for full activation of the endonuclease but insufficient for multiple turnovers of the methyltransferase. The mode of action of BsgI on DNA with two recognition sites is, however, not unique to this particular subset of type IIs enzymes, as the same mode was observed with Acc36I (Table II). Yet Acc36I is an isoschizomer of BspMI, an enzyme that behaves in a radically different manner (see later and accompanying paper (36)).

Scheme C, FokI-- A third mode of action was noted with FokI (Fig. 5). Like BsgI, FokI cleaved the plasmid with two copies of its recognition site more rapidly than plasmids with a single copy. The two single-site plasmids for FokI, carrying either one of the sites present in the two-site DNA, were both cleaved slowly so the enhanced rate on the DNA with two sites must be due to the presence of two FokI sites in the same molecule of DNA. The optimal activity of FokI therefore involves a bridging interaction between two copies of its recognition sequence in cis. However, during the course of the FokI reaction on the DNA with two sites (Fig. 5b), the FLL DNA cleaved at one site accumulated to a higher level than in the equivalent reaction with BsgI (Fig. 3b). The interaction of FokI with two DNA sites in cis thus results in the rapid cleavage of just one site. The other site presumably functions as an activator for DNA cleavage rather than as a second substrate, in much the same way as with the type IIe restriction enzymes. Indeed, the reaction profile of FokI on its two-site substrate is very similar to that for the type IIe enzyme NaeI (22).

One scheme for how the monomeric FokI protein might interact concurrently with two DNA sites is that a monomer binds separately to each site and that two monomers then associate to form the dimer necessary for the DNA cleavage reaction (28, 31, 32). However, if this were the case, FokI should cleave a two-site substrate faster than a one-site substrate only when the enzyme is present at a higher concentration than the DNA. Otherwise, only a small fraction of the DNA will carry two monomers of the protein at the same time. For example, with the enzyme at a 10-fold lower concentration than the DNA (as in Fig. 5), then 1% of the DNA bound to enzyme will carry two molecules of the enzyme. Yet, in contrast to this scheme, enhanced activity on the two-site substrate was observed here in reactions with excess DNA over protein. Moreover, the rates of cleavage of the two-site substrate varied non-linearly with the concentration of FokI in the same manner as those for the cleavage of the one-site substrate (Fig. 6). This indicates that the assembly of the active dimeric form of FokI occurs by the same pathway on one-site and two-site substrates.

Rather than an association between two monomers that are each bound to a separate recognition site, an alternative pathway is that a monomer of FokI bound to one recognition site recruits a second monomer from free solution. The crystal structure of the free FokI protein shows a rather small dimer interface (31). The complex containing two monomers at a single recognition site may therefore be relatively unstable, and it may often fall apart before cleaving the DNA, thus resulting in a relatively slow reaction velocity on a DNA with one site. On the other hand, if the DNA has two sites, the monomer recruited to the DNA-protein complex at one site will be able to bind to the second site via its DNA-recognition domain and thus stabilize the protein-protein association, in the same way as proposed for the  repressor (34). The complex spanning two sites may seldom fall apart before cleaving the DNA, thus resulting in a much faster reaction velocity on a DNA with two sites. In contrast to the pathway involving the association of two DNA-bound monomers, this alternative scheme can account for the variation in the cleavage rates on a two-site substrate with the FokI concentration (Fig. 6b), but it requires that the FokI monomer has a higher affinity for the protein bound to the recognition site than for the vacant recognition site in the same DNA. Whether this is so has yet to be determined.

Scheme D, BspMI-- The fourth mode of action was observed with BspMI (Fig. 7). As with BsgI and FokI, BspMI acted much more rapidly on the plasmid with two copies of its recognition site than on that with one copy, but BspMI gives a different reaction profile on its two-site substrate from either BsgI or FokI. The major product(s) liberated from the BspMI protein during its reaction on the DNA with two BspMI sites are the fragments cut at both sites in both strands: Only a small fraction was liberated from the enzyme as FLL DNA cut at one site. Hence, the binding of BspMI to a DNA with two target sites generally results in the cleavage of all four of the scissile phosphodiester bonds, two at each target, before the enzyme dissociates from the DNA, although it occasionally dissociates after cutting two bonds. The profile of the steady-state reaction of BspMI on its two-site substrate is thus similar to those of the tetrameric type IIf restriction endonucleases, such as SfiI, Cfr10I, and NgoMIV (18-22). Further studies, described in the accompanying paper (36), show that BspMI is a tetramer that has to bind to two copies of its recognition sequence before cleaving DNA.

This work has shown that the type IIs restriction enzymes display considerable diversity in the mechanisms by which they cleave their DNA substrates. This is in contrast to the orthodox type II restriction enzymes that are often similar to one another in terms of structure and/or reaction mechanism: viz. EcoRV and BglI (4, 6, 8, 9), and EcoRI and BamHI (7). Moreover, although some of the type II restriction enzymes that recognize palindromic DNA sequences have to interact with two copies of the relevant recognition sequence before they can cleave DNA (11, 12), the requirement for two sites seems to be particularly common, although not universal, among the type IIs enzymes. Other type IIs enzymes that have recently been shown to need two sites include MboII (48), which follows scheme B, like BsgI (Fig. 3), and BfiI,⁴ that yields reaction profiles similar to those for scheme C, like FokI (Fig. 5). The type I and III restriction enzymes also need two copies of their recognition sites for most or all of their reactions (49). Hence, a substantial fraction, perhaps the majority, of the restriction enzymes that exist in nature are endonucleases that have to interact with two sites before cleaving the DNA. The need for two sites can enhance the ability of these enzymes to discriminate between cognate and non-cognate DNA and to ensure that they cleave only the former (23).

	ACKNOWLEDGEMENTS

We thank Jurate Bitinaite, Ira Schildkraut, Huimin Kong, and Geoff Wilson for advice and reagents; Bernard Connolly and Virginijus Siksnys for pre-publication data; and Mark Szczelkun and Lucy Daniels for comments.

	FOOTNOTES

^* This work was supported by grants 7/G10881 from the Biotechnology and Biological Sciences Research Council and 046178 from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-117-928-7429; Fax: 44-117-928-8274; E-mail: s.halford@bris.ac.uk.

Published, JBC Papers in Press, November 29, 2001, DOI 10.1074/jbc.M108441200

² H. Kong (New England BioLabs), personal communication.

³ G. Wilson (New England BioLabs), personal communication.

⁴ G. Sasnauskas and V. Siksnys (Institute of Biotechnology, Vilnius), personal communication.

	ABBREVIATIONS

The abbreviations used are: AdoMet, S-adenosyl methionine; DTT, dithiothreitol; BSA, bovine serum albumin; SC, supercoiled; OC, open-circle; FLL, full-length linear; L1 and L2, linear DNA fragments from cutting a circular DNA with two sites at both sites.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				G. Sasnauskas, S. E. Halford, and V. Siksnys How the BfiI restriction enzyme uses one active site to cut two DNA strands PNAS, May 27, 2003; 100(11): 6410 - 6415. [Abstract] [Full Text] [PDF]

				N. A. Gormley, A. L. Hillberg, and S. E. Halford The Type IIs Restriction Endonuclease BspMI Is a Tetramer That Acts Concertedly at Two Copies of an Asymmetric DNA Sequence J. Biol. Chem., February 1, 2002; 277(6): 4034 - 4041. [Abstract] [Full Text] [PDF]

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