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

2002; Elsevier BV; Volume: 277; Issue: 6 Linguagem: Inglês

10.1074/jbc.m108441200

1083-351X

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

Advanced biosensing and bioanalysis techniques

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. 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. S-adenosyl methionine dithiothreitol bovine serum albumin supercoiled open-circle full-length linear linear DNA fragments from cutting a circular DNA with two sites at both sites Over 3000 type II restriction endonucleases have been identified to date (1Roberts R.J. Macelis D. Nucleic Acids Res. 2001; 29: 268-269Crossref PubMed Scopus (115) Google Scholar). 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 (2Roberts R.J. Halford S.E. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 35-88Google Scholar). With one exception, BfiI (3Sapranauskas R. Sasnauskas D. Lagunavicius A. Vilkaitis G. Lubys A. Siksnys V. J. Biol. Chem. 2000; 275: 30878-30885Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), they require Mg2+ or a similar divalent metal ion to cleave DNA (4Baldwin G.S. Sessions R.B. Erskine S.G. Halford S.E. J. Mol. Biol. 1999; 288: 87-104Crossref PubMed Scopus (69) Google Scholar), although a few also require AdoMet1 for maximal activity (5Janulaitis A. Petrusyte M. Maneliene Z. Klimasauskas S. Butkus V. Nucleic Acids Res. 1992; 20: 6043-6049Crossref PubMed Scopus (63) Google Scholar). 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 (6Winkler F.K. Banner D.W. Oefner C. Tsernoglou D. Brown R.S. Heathman S.P. Bryan R.K. Martin P.D. Petratos K. Wilson K.S. EMBO J. 1993; 12: 1781-1795Crossref PubMed Scopus (442) Google Scholar, 7Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar, 8Newman M. Lunnen K. Wilson G. Greci J. Schildkraut I. Phillips S.E.V. EMBO J. 1998; 17: 5466-5476Crossref PubMed Scopus (133) Google Scholar). Enzymes of this sort cleave DNA with multiple target sites by means of separate reactions at each site (9Gormley N.A. Bath A.J. Halford S.E. J. Biol. Chem. 2000; 275: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), although they can act processively and migrate from one site to another by intramolecular processes (10Stanford N.P. Szczelkun M.D. Marko J.F. Halford S.E. EMBO J. 2000; 19: 6546-6557Crossref PubMed Scopus (153) Google Scholar).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 (11Halford S.E. Bilcock D.T. Stanford N.P. Williams S.A. Milsom S.E. Gormley N.A. Watson M.A. Bath A.J. Embleton M.L. Gowers D.M. Daniels L.E. Parry S.H. Szczelkun M.D. Biochem. Soc. Trans. 1999; 27: 696-699Crossref PubMed Scopus (24) Google Scholar, 12Halford S.E. Biochem. Soc. Trans. 2001; 29: 363-373Crossref PubMed Scopus (57) Google Scholar). The latter include the type IIe enzymes, such as EcoRII,NaeI, and Sau3AI (13Krüger D.H. Barcak G.J. Reuter M. Smith H.O. Nucleic Acids Res. 1988; 16: 3997-4008Crossref PubMed Scopus (115) Google Scholar, 14Oller A.R. Vanden Broek W. Conrad M. Topal M.D. Biochemistry. 1991; 30: 2543-2549Crossref PubMed Scopus (59) Google Scholar, 15Reuter M. Kupper D. Meisel A. Schroeder C. Krüger D.H. J. Biol. Chem. 1998; 273: 8294-8300Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Sruct. Biol. 2000; 8: 665-669Crossref Scopus (46) Google Scholar, 17Friedhoff P. Lurz R. Lüder G. Pingoud A. J. Biol. Chem. 2001; 271: 23581-23588Abstract Full Text Full Text PDF Scopus (53) Google Scholar), and the type IIf enzymes, such as SfiI, SgrAI, Cfr10I, and NgoMIV (18Wentzell L.M. Nobbs T.J. Halford S.E. J. Mol. Biol. 1995; 248: 581-595Crossref PubMed Scopus (110) Google Scholar, 19Bilcock D.T. Daniels L.E. Bath A.J. Halford S.E. J. Biol. Chem. 1999; 274: 36379-36386Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Siksnys V. Skirgaila R. Sasnauskas G. Urbanke C. Cherny D. Grazulis S. Huber R. J. Mol. Biol. 1999; 291: 1105-1118Crossref PubMed Scopus (78) Google Scholar, 21Deibert M. Grazulis S. Sasnauskas G. Siksnys V. Huber R. Nat. Struct. Biol. 2000; 7: 792-799Crossref PubMed Scopus (146) Google Scholar). 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 (22Embleton M.L. Siksnys V. Halford S.E J. Mol. Biol. 2001; 311: 503-515Crossref PubMed Scopus (65) Google Scholar). Except for Sau3AI, a monomer in free solution (17Friedhoff P. Lurz R. Lüder G. Pingoud A. J. Biol. Chem. 2001; 271: 23581-23588Abstract Full Text Full Text PDF Scopus (53) Google Scholar), 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 (15Reuter M. Kupper D. Meisel A. Schroeder C. Krüger D.H. J. Biol. Chem. 1998; 273: 8294-8300Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Sruct. Biol. 2000; 8: 665-669Crossref Scopus (46) Google Scholar). In contrast, the type IIf enzymes are generally tetramers with two identical surfaces for binding their palindromic sites, each made from two subunits (18Wentzell L.M. Nobbs T.J. Halford S.E. J. Mol. Biol. 1995; 248: 581-595Crossref PubMed Scopus (110) Google Scholar, 21Deibert M. Grazulis S. Sasnauskas G. Siksnys V. Huber R. Nat. Struct. Biol. 2000; 7: 792-799Crossref PubMed Scopus (146) Google Scholar).The type II family contains another subgroup, the type IIs systems (24Szybalski W. Kim S.C. Hasan N. Podhajska A.J. Gene (Amst.). 1991; 100: 13-26Crossref PubMed Scopus (197) Google Scholar). 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 (1Roberts R.J. Macelis D. Nucleic Acids Res. 2001; 29: 268-269Crossref PubMed Scopus (115) Google Scholar). Several hundred type IIs enzymes have been identified, and those characterized to date are monomers in solution (25Kaczorowski T. Skowron P. Podhajska A.J. Gene (Amst.). 1989; 80: 209-216Crossref PubMed Scopus (45) Google Scholar, 26Sektas M. Kaczorowski T. Podajska A.J. Nucleic Acids Res. 1992; 20: 433-438Crossref PubMed Scopus (19) Google Scholar, 27Tucholski J. Skowron P. Podjhaska A.J. Gene (Amst.). 1995; 157: 87-92Crossref PubMed Scopus (28) Google Scholar, 28Higgins L.S. Besnier C. Kong H. Nucleic Acids Res. 2001; 29: 2492-2501Crossref PubMed Google Scholar). 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 (29Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar). 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 (30Wah D.A. Hirsh J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). 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 (31Wah D.A. Bitinaite J. Schildkraut I. Aggarwal A.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10564-10569Crossref PubMed Scopus (174) Google Scholar). 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 (32Bitinaite J. Wah D.A. Aggarwal A.K. Schildkraut I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10570-10575Crossref PubMed Scopus (377) Google Scholar). 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 (32Bitinaite J. Wah D.A. Aggarwal A.K. Schildkraut I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10570-10575Crossref PubMed Scopus (377) Google Scholar). 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 (33Vanamee E.S. Santagata S. Aggarwal A.K. J. Mol. Biol. 2001; 309: 69-78Crossref PubMed Scopus (140) Google Scholar). Nevertheless, the pathway for the assembly of aFokI dimer at its recognition site(s) has yet to be established. The monomer bound to the recognition sequence (30Wah D.A. Hirsh J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar) 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 (31Wah D.A. Bitinaite J. Schildkraut I. Aggarwal A.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10564-10569Crossref PubMed Scopus (174) Google Scholar, 32Bitinaite J. Wah D.A. Aggarwal A.K. Schildkraut I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10570-10575Crossref PubMed Scopus (377) Google Scholar). DNA cleavage by FokI may thus require a bridging interaction between two copies of its recognition sequence (33Vanamee E.S. Santagata S. Aggarwal A.K. J. Mol. Biol. 2001; 309: 69-78Crossref PubMed Scopus (140) Google Scholar). 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(34Hochschild A. Cozzarelli N.R. Wang J.C. DNA Topology and Its Biological Effects. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990: 107-138Google Scholar).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 oneBspMI site, but its cleavage of this plasmid is stimulated by the addition of DNA molecules that have multiple BspMI sites (14Oller A.R. Vanden Broek W. Conrad M. Topal M.D. Biochemistry. 1991; 30: 2543-2549Crossref PubMed Scopus (59) Google Scholar). Similarly, Eco57I can be stimulated to cleave refractory sites by the addition of an oligonucleotide duplex that carries its recognition sequence (35Reuter M. Kupper D. Pein C.-D. Petrusyte M. Siksnys V. Frey B. Krüger D.H. Anal. Biochem. 1993; 209: 232-237Crossref PubMed Scopus (34) Google Scholar). 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 (13Krüger D.H. Barcak G.J. Reuter M. Smith H.O. Nucleic Acids Res. 1988; 16: 3997-4008Crossref PubMed Scopus (115) Google Scholar, 14Oller A.R. Vanden Broek W. Conrad M. Topal M.D. Biochemistry. 1991; 30: 2543-2549Crossref PubMed Scopus (59) Google Scholar, 15Reuter M. Kupper D. Meisel A. Schroeder C. Krüger D.H. J. Biol. Chem. 1998; 273: 8294-8300Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Huai Q. Colandene J.D. Topal M.D. Ke H. Nat. Sruct. Biol. 2000; 8: 665-669Crossref Scopus (46) Google Scholar, 17Friedhoff P. Lurz R. Lüder G. Pingoud A. J. Biol. Chem. 2001; 271: 23581-23588Abstract Full Text Full Text PDF Scopus (53) Google Scholar, 18Wentzell L.M. Nobbs T.J. Halford S.E. J. Mol. Biol. 1995; 248: 581-595Crossref PubMed Scopus (110) Google Scholar, 19Bilcock D.T. Daniels L.E. Bath A.J. Halford S.E. J. Biol. Chem. 1999; 274: 36379-36386Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Siksnys V. Skirgaila R. Sasnauskas G. Urbanke C. Cherny D. Grazulis S. Huber R. J. Mol. Biol. 1999; 291: 1105-1118Crossref PubMed Scopus (78) Google Scholar, 21Deibert M. Grazulis S. Sasnauskas G. Siksnys V. Huber R. Nat. Struct. Biol. 2000; 7: 792-799Crossref PubMed Scopus (146) Google Scholar, 22Embleton M.L. Siksnys V. Halford S.E J. Mol. Biol. 2001; 311: 503-515Crossref PubMed Scopus (65) Google Scholar).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 (19Bilcock D.T. Daniels L.E. Bath A.J. Halford S.E. J. Biol. Chem. 1999; 274: 36379-36386Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (12Halford S.E. Biochem. Soc. Trans. 2001; 29: 363-373Crossref PubMed Scopus (57) Google Scholar). 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 (22Embleton M.L. Siksnys V. Halford S.E J. Mol. Biol. 2001; 311: 503-515Crossref PubMed Scopus (65) Google Scholar). The accompanying paper (36Gormley N.A. Hillberg A. Halford S.E. J. Biol. Chem. 2002; 277: 4034-4041Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) 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.RESULTSThe 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 TableI. 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 (2Roberts R.J. Halford S.E. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 35-88Google Scholar), 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).Table IEnzymes and substratesEnzymeSpecies of originRecognition site1-aThe first andsecond figures in the parentheses denote the number of bp between the 3′ end of the recognition sequence and the points of cleavage in, respectively, the "top" and "bottom" strands.One-site plasmidTwo-site plasmidDNA between sitesFlanking identity1-bThe first andsecond figures denote the number of bp of identical sequence flanking each recognition site on, respectively, the 5′ and 3′ sides.bpBsmIBacillus stearothermophilusNUB36GAATGC(1/−1)pBR322pML213572 /2BsmBIBacillus stearothermophilusB61CGTCTC(1/5)pBR322pML221342 /2BsaIBacillus stearothermophilus6–55GGTCTC(1/5)pBR322pML29572 /3SapISaccharopolysporaspeciesGCTCTTC(1/4)pBR322pML220302 /3BsgIBacillus sphaericusGTGCAG(16/14)pBR322pML216282 /2BpmIBacillus pumillusCTGGAG(16/14)pNEB193pAB513153 /18FokIFlavobacterium okeonokoitesGGATG(9/13)pSKFok1 and pSKFok3pSKFok21895 /15BspMIBacillus species MACCTGC(4/8)pAT153pNAG17009 /15Acc36IAcinetobacter calcoaceticus36ACCTGC(4/8)pAT153pNAG17009 /15The name, source, and recognition sequence for the type IIs restriction enzymes examined are listed, along with the plasmids that were used as substrates for each enzyme. On the plasmids with two sites, the length of DNA between the sites is noted, as is the length of flanking DNA sequences on either side of each site that are identical at both sites.1-a The first andsecond figures in the parentheses denote the number of bp between the 3′ end of the recognition sequence and the points of cleavage in, respectively, the "top" and "bottom" strands.1-b The first andsecond figures denote the number of bp of identical sequence flanking each recognition site on, respectively, the 5′ and 3′ sides. Open table in a new tab 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 (40Huang J. Schlick T. Vologodskii A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 968-973Crossref PubMed Scopus (83) Google Scholar, 41Milsom S.E. Halford S.E. Embleton M.L. Szczelkun M.D. J. Mol. Biol. 2001; 311: 517-528Crossref Scopus (31) Google Scholar). 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 (42Stark W.M. Boocock M.A. Sherratt D.J. Mobile Genetic Elements. Oxford University Press, Oxford, UK1995: 101-129Google Scholar). 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 SapIThese 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).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 twoBsmBI 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 (9Gormley N.A. Bath A.J. Halford S.E. J. Biol. Chem. 2000; 275: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Bilcock D.T. Daniels L.E. Bath A.J. Halford S.E. J. Biol. Chem. 1999; 274: 36379-36386Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (TableII).Table IIReaction rates on one-site and two-site substratesEnzymeEnzyme concentrationReaction bufferReaction temperatureRate of utilization of one-site DNARate of utilization of two-site DNARatio of rates: two-site DNA/one-site DNARel. rates on two-site DNA (first site/second site)Reaction scheme°Cnm/minBsmI12 units/mlB650.230.261.1∼1ABsmBI48 units/mlC550.380.461.2∼1ABsaI24 units/mlA500.110.131.2∼1ASapI12 units/mlA371.31.81.4∼1ABsgI1.2 units/mlA+370.0791.924∼1BBpmI10 units/mlC370.0811.417∼1BFokI1 nmA370.0252-aOf the two values cited, the upper refers to pSKFok1 and the lower to pSKFok3.0.36142-aOf the two values cited, the upper refers to pSKFok1 and the lower to pSKFok3.≫1C0.03111BspMI0.25 nmD370.0080.1721≪1DAcc36I30 units/mlD370.0040.1128∼1BThe reactions contained the enzyme at the concentration noted (units/ml for enzymes from commercial suppliers; nm for pure proteins) in 200 μl of buffer at the indicated temperature, and 5 nm DNA (except for FokI and Acc36I, with 10 nm DNA). The DNA had either one or two copies of the recognition sequence for the enzyme in question (Table I). Samples were withdrawn from the reactions at various times (0–240 min) and analyzed as in "Experimental Procedures" to determine the initial rates for the decline in the concentration of the supercoiled substrates (nm DNA consumed/min). The rates on both substrates are noted here, as are the ratios of the rates on the two-site over the one-site DNA and the relative rates for cutting each site in the two-site substrates. The reaction schemes, A–D, are as noted under "Discussion."2-a Of the two values cited, the upper refers to pSKFok1 and the lower to pSKFok3. Open table in a new tab 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 (12Halford S.E. Biochem. Soc. Trans. 2001; 29: 363-373Crossref PubMed Scopus (57) Google Scholar)), 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 (9Gormley N.A. Bath A.J. Halford S.E. J. Biol. Chem. 2000; 275: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,19Bilcock D.T. Daniels L.E. Bath A.J. Halford S.E. J. Biol. Chem. 1999; 274: 36379-36386Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).BsmBI, BsmI, BsaI, and SapI all cleave DNA in the manner of an orthodox type II restriction enzyme, such as EcoRV, BamHI, and BglI (9Gormley N.A. Bath A.J. Halford S.E. J. Biol. Chem. 2000; 275: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). 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 BpmIIn 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 (43Wilson G.G. Nucleic Acids Res. 1991; 19: 2539-2565Crossref PubMed Scopus (199) Google Scholar), but Eco57I differs from most type IIs systems in that a single polypeptide carries both the endonuclease and one of the methyltransferase activities (44Janulaitis A. Vaisvila R. Timinskas A. Klimasauskas S. Butkus V. Nucleic Acids Res. 1992; 20: 6051-6056Crossref PubMed Scopus (52) Google Scholar). 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 (5Janulaitis A. Petrusyte M. Maneliene Z. Klimasauskas S. Butkus V. Nucleic Acids Res. 1992; 20: 6043-6049Crossref PubMed Scopus (63) Google Scholar). BsgI and BpmI have similar genetic organizations and homologous amino acid sequences to Eco57I. 2H. Kong (New England BioLabs), personal communication. DNA cleavage by BsgI is also stimulated by AdoMet,2although BpmI has yet to be tested in this regard (1Roberts R.J. Macelis D. Nucleic Acids Res. 2001; 29: 268-269Crossref PubMed Scopus (115) Google Scholar).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 wa