Reactions of BglI and Other Type II Restriction Endonucleases with Discontinuous Recognition Sites
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.6928
ISSN1083-351X
AutoresNiall Gormley, Abigail J. Bath, Stephen E. Halford,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoType II restriction enzymes generally recognize continuous sequences of 4–8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA.BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV.BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism. Type II restriction enzymes generally recognize continuous sequences of 4–8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA.BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV.BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism. base pair(s) dithiothreitol supercoiled open circle full-length linear linear DNA fragments from the cleavage of a circular DNA with two sites at both sites Type II restriction endonucleases recognize specific sequences in DNA and cut both strands at fixed locations within or adjacent to the recognition sequence, in reactions that need only Mg2+ as a cofactor (1.Roberts 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). The value of these enzymes as tools for the analysis and manipulation of DNA has prompted extensive searches for new enzymes of this type, and over 3000 have been identified (2.Roberts R.J. Macelis D. Nucleic Acids Res. 1999; 26: 312-313Crossref Scopus (39) Google Scholar). The recognition sites for most restriction enzymes are palindromic sequences of 4, 6, or 8 consecutive bp.1 Almost all of the relatively small number of type II enzymes that have been analyzed to date, with respect to their reaction mechanisms and/or structures, recognize continuous sequences of this sort: viz. BamHI, EcoRI, EcoRV, MunI,PvuII, and TaqI (3.Dorner L.F. Bitinaite J. Whitaker R.D. Schildkraut I. J. Mol. Biol. 1999; 285: 1515-1523Crossref PubMed Scopus (31) Google Scholar, 4.Lesser D.R. Kurpiewski M.R. Waters T. Connolly B.A. Jen-Jacobson L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7548-7552Crossref PubMed Scopus (85) Google Scholar, 5.Baldwin G.S. Sessions R.B. Erskine S.G. Halford S.E. J. Mol. Biol. 1999; 288: 87-104Crossref PubMed Scopus (69) Google Scholar, 6.Deibert M. Grazulis S. Janulaitis A. Siksnys V. Huber R. EMBO J. 1999; 21: 5805-5816Crossref Scopus (67) Google Scholar, 7.Nastri H.G. Evans P.D. Walker I.H. Riggs P.D. J. Biol. Chem. 1997; 272: 25761-25767Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8.Cao W. Biochemistry. 1999; 38: 8080-8087Crossref PubMed Scopus (8) Google Scholar). All of these examples are homodimeric proteins that interact symmetrically with their respective recognition sites so that the two active sites are located on the two target phosphodiester bonds, one in each strand. The enzyme can then cut both strands at one site in a single DNA-binding event (9.Erskine S.G. Baldwin G.S. Halford S.E. Biochemistry. 1997; 36: 7567-7576Crossref PubMed Scopus (64) Google Scholar, 10.Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). On a DNA with two or more sites, they usually act in a distributive manner (11.Bilcock 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), catalyzing separate reactions at each site, but they sometimes act processively, translocating to a second site and cutting it before leaving the DNA (10.Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 12.Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar). With a few exceptions, the type II restriction enzymes have dissimilar amino acid sequences (13.Wilson G.G. Murray N.E. Annu. Rev. Genet. 1991; 25: 585-627Crossref PubMed Scopus (586) Google Scholar) but their three-dimensional structures can be similar to one another (14.Aggarwal A.K. Curr. Opin. Struct. Biol. 1995; 5: 11-19Crossref PubMed Scopus (172) Google Scholar). For instance, the tertiary fold ofEcoRI is very similar to BamHI (15.Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1994; 368: 660-664Crossref PubMed Scopus (161) Google Scholar); likewiseEcoRV is similar to PvuII (16.Kostrewa D. Winkler F.K. Biochemistry. 1995; 34: 683-696Crossref PubMed Scopus (241) Google Scholar), though the overall structures of EcoRI and EcoRV differ considerably. The mechanisms of these enzymes also show similarities in some instances and differences in other instances. For example,EcoRI and BamHI bind to DNA in the absence of Mg2+ preferentially at their recognition sites (3.Dorner L.F. Bitinaite J. Whitaker R.D. Schildkraut I. J. Mol. Biol. 1999; 285: 1515-1523Crossref PubMed Scopus (31) Google Scholar, 4.Lesser D.R. Kurpiewski M.R. Waters T. Connolly B.A. Jen-Jacobson L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7548-7552Crossref PubMed Scopus (85) Google Scholar, 12.Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar). In contrast, under their optimal reaction conditions but for the absence of Mg2+, EcoRV, TaqI, and several others bind equally well to all DNA sequences (17.Taylor J.D. Badcoe I.G. Clarke A.R. Halford S.E. Biochemistry. 1991; 30: 8743-8753Crossref PubMed Scopus (145) Google Scholar, 18.Zebala J.F. Choi J. Barany F. J. Biol. Chem. 1992; 267: 8097-8105Abstract Full Text PDF PubMed Google Scholar, 19.Lagunavicius A. Grazulis S. Balciunaite E. Vainius D. Siksnys V. Biochemistry. 1997; 37 (11099): 11903Google Scholar, 20.Erskine S.G. Halford S.E. J. Mol. Biol. 1998; 275: 759-772Crossref PubMed Scopus (44) Google Scholar). In the presence of Mg2+, the latter enzymes still cleave DNA specifically at their recognition sites (21.Taylor J.D. Halford S.E. Biochemistry. 1989; 28: 6198-6207Crossref PubMed Scopus (119) Google Scholar, 22.Zebala J.F. Choi J. Trainor J. Barany F. J. Biol. Chem. 1992; 267: 8106-8116Abstract Full Text PDF PubMed Google Scholar), but they also need a divalent metal ion for specific binding; Ca2+ can function in this respect (7.Nastri H.G. Evans P.D. Walker I.H. Riggs P.D. J. Biol. Chem. 1997; 272: 25761-25767Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8.Cao W. Biochemistry. 1999; 38: 8080-8087Crossref PubMed Scopus (8) Google Scholar, 23.Vipond I.B. Halford S.E. Biochemistry. 1995; 34: 1113-1119Crossref PubMed Scopus (135) Google Scholar, 24.Martin A.M. Horton N.C. Lusetti S. Reich N.O. Perona J.J. Biochemistry. 1999; 38: 8430-8439Crossref PubMed Scopus (64) Google Scholar). The similarities and the differences among the type II restriction enzymes have led to the proposal that many, including BamHI, can be classified asEcoRI-like enzymes, whereas many others, includingPvuII and TaqI, can be classified asEcoRV-like enzymes (10.Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14.Aggarwal A.K. Curr. Opin. Struct. Biol. 1995; 5: 11-19Crossref PubMed Scopus (172) Google Scholar, 20.Erskine S.G. Halford S.E. J. Mol. Biol. 1998; 275: 759-772Crossref PubMed Scopus (44) Google Scholar, 22.Zebala J.F. Choi J. Trainor J. Barany F. J. Biol. Chem. 1992; 267: 8106-8116Abstract Full Text PDF PubMed Google Scholar). However, an alternative view maintains that there is no clear-cut distinction between theEcoRI- and the EcoRV-like enzymes and that these enzymes are essentially similar to each other in terms of both mechanism and core structure (6.Deibert M. Grazulis S. Janulaitis A. Siksnys V. Huber R. EMBO J. 1999; 21: 5805-5816Crossref Scopus (67) Google Scholar, 19.Lagunavicius A. Grazulis S. Balciunaite E. Vainius D. Siksnys V. Biochemistry. 1997; 37 (11099): 11903Google Scholar, 24.Martin A.M. Horton N.C. Lusetti S. Reich N.O. Perona J.J. Biochemistry. 1999; 38: 8430-8439Crossref PubMed Scopus (64) Google Scholar, 25.Engler L.E. Welch K.K. Jen-Jacobson L. J. Mol. Biol. 1997; 269: 82-101Crossref PubMed Scopus (96) Google Scholar, 26.Pingoud A. Jeltsch A. Eur. J. Biochem. 1997; 246: 1-22Crossref PubMed Scopus (302) Google Scholar). The recognition sites for some type II restriction enzymes are not the continuous sequences of 4–8 consecutive bp noted above, but are instead discontinuous sequences in which the palindromic elements recognized by the enzyme are separated by a fixed length of nonspecific DNA (2.Roberts R.J. Macelis D. Nucleic Acids Res. 1999; 26: 312-313Crossref Scopus (39) Google Scholar). The intervening DNA can be as long as 9 bp, viz. XcmI (Table I), or as short as 1 bp, viz. HhaII (GANTC). Apart from some early studies on HhaII (27.Kaddurah-Daouk R. Cho P. Smith H.O. J. Biol. Chem. 1985; 260: 15345-15351Abstract Full Text PDF PubMed Google Scholar),SfiI is the only type II enzyme with a discontinuous recognition site whose reaction mechanism has been analyzed (28.Wentzell L.M. Nobbs T.J. Halford S.E. J. Mol. Biol. 1995; 248: 581-595Crossref PubMed Scopus (110) Google Scholar, 29.Nobbs T.J. Halford S.E. J. Mol. Biol. 1995; 252: 399-411Crossref PubMed Scopus (39) Google Scholar, 30.Szczelkun M.D. Halford S.E. EMBO J. 1996; 15: 1460-1469Crossref PubMed Scopus (54) Google Scholar, 31.Nobbs T.J. Szczelkun M.D. Wentzell L.M. Halford S.E. J. Mol. Biol. 1998; 281: 419-432Crossref PubMed Scopus (56) Google Scholar, 32.Wentzell L.M. Halford S.E. J. Mol. Biol. 1998; 281: 433-444Crossref PubMed Scopus (58) Google Scholar, 33.Embleton M.L. Williams S.A. Watson M.A. Halford S.E. J. Mol. Biol. 1999; 289: 785-797Crossref PubMed Scopus (50) Google Scholar).SfiI differs from the orthodox enzymes such asEcoRV in several ways. First, its recognition site contains 8 specified bp interrupted by 5 bp of nonspecific DNA and thus covers 13 bp (Table I), which is longer than usual for a restriction site (34.Qiang B-Q. Schildkraut I. Nucleic Acids Res. 1984; 12: 4507-4516Crossref PubMed Scopus (59) Google Scholar). Second, SfiI is a tetramer of identical subunits instead of the normal dimer. Third, no DNA cleavage arises from the binding of the tetramer to one recognition site. Instead,SfiI is only active when bound to two copies of its site. It displays its optimal activity with two sites on the same DNA, where it loops out the DNA between the sites; but it can also, albeit less readily, cleave DNA with one SfiI site by bridging two such molecules. Fourth, once bound to the two sites, SfiI usually cuts both strands at both sites before leaving the DNA. Another subset of the type II enzymes, known as type IIe and typified byEcoRII and NaeI, also needs two sites for the DNA cleavage reactions but these differ from SfiI in that they seem to use one site to activate the reaction at the other site; the activator DNA is not cleaved (35.Yang C.C. Topal M.D. Biochemistry. 1992; 31: 9657-9664Crossref PubMed Scopus (44) Google Scholar, 36.Gabbara S. Bhagwat A.S. J. Biol. Chem. 1992; 267: 18623-18630Abstract Full Text PDF PubMed Google Scholar, 37.Reuter 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). Nonetheless, concerted cleavage of two recognition sites in the manner of SfiI has been noted with both Cfr10I and SgrAI (11.Bilcock 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, 38.Siksnys 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). UnlikeSfiI, Cfr10I and SgrAI recognize continuous sequences, R↓CCGGY and GR↓CCGGYC, respectively. The 8-bp site for SgrAI is identical to the 6-bp site forCfr10I except for one extra bp at each end.Table IRecognition sequencesEnzymeSpecies of originRecognition sequencea↓ marks the point of cleavage and N denotes any base.SfiIStreptomyces fimbriatusGGCCNNNN↓NGGCCBglIBacillus globigiiGCCNNNN↓NGGCTth111IThermus thermophilus 111GACN↓NNGTCPshAIPlesiomonas shigelloidesGACNN↓NNGTCAhdIAeromonas hydophiliaGACNNNN↓NNGTCDrdIDeinococcus radioduransGACNNNNN↓NNGTCPflMIPseudomonas fluorescencesCCANNNN↓NTGGBstXIBacillus stearothermophilusXCCANNNNN↓NTGGXcmIXanthamonas campestrisCCANNNNN↓NNNNTGGThe name, the species of origin, and the recognition sequence (2.Roberts R.J. Macelis D. Nucleic Acids Res. 1999; 26: 312-313Crossref Scopus (39) Google Scholar) for all of the restriction enzymes examined here are listed. The enzymes are grouped on the basis of similarities in their recognition sequences.a ↓ marks the point of cleavage and N denotes any base. Open table in a new tab The name, the species of origin, and the recognition sequence (2.Roberts R.J. Macelis D. Nucleic Acids Res. 1999; 26: 312-313Crossref Scopus (39) Google Scholar) for all of the restriction enzymes examined here are listed. The enzymes are grouped on the basis of similarities in their recognition sequences. In this study, the reactions of BglI (39.Lee Y.H. Chirikjian J.G. J. Biol. Chem. 1979; 254: 6838-6841Abstract Full Text PDF PubMed Google Scholar) and several other restriction enzymes with discontinuous recognition sites (Table I) were analyzed on plasmids with either one or two target sites, to determine whether enzymes with elongated sites behave like the orthodox enzymes or whether they interact with two sites, like SfiI. The recognition sequence for BglI (40.Bickle T.A. Ineichen K. Gene (Amst.). 1980; 9: 205-212Crossref PubMed Scopus (21) Google Scholar) is identical to that forSfiI except that it is one bp shorter at each end (Table I),i.e. the same relationship as that between theCfr10I and SgrAI sites. This raises the possibility that BglI might act concertedly at two sites in the same way as SfiI. However, the structure ofBglI bound to its recognition sequence was recently determined by x-ray crystallography, and this shows a dimeric protein bound symmetrically to one DNA duplex (41.Newman M. Lunnen K. Wilson G. Greci J. Schildkraut I. Phillips S.E.V. EMBO J. 1998; 17: 5466-5476Crossref PubMed Scopus (134) Google Scholar). Moreover, the DNA recognition and catalytic functions in each subunit of BglI are similar to those in EcoRV, though the subunit interface in BglI differs from EcoRV. The amino acid sequences of BglI and EcoRV are not homologous (13.Wilson G.G. Murray N.E. Annu. Rev. Genet. 1991; 25: 585-627Crossref PubMed Scopus (586) Google Scholar). In EcoRV, which cleaves GAT↓ATC as marked, the recognition/catalytic functions of the two subunits are located almost opposite each other across the axis of the DNA (42.Winkler 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: 1781-17945Crossref PubMed Scopus (445) Google Scholar). In contrast, the recognition/catalytic functions of the two subunits in BglI are displaced relative to each other along the axis of the DNA, thus allowing it to recognize two specific segments of DNA separated by 5 bp of nonspecific DNA (41.Newman M. Lunnen K. Wilson G. Greci J. Schildkraut I. Phillips S.E.V. EMBO J. 1998; 17: 5466-5476Crossref PubMed Scopus (134) Google Scholar). The main question posed here is whetherBglI acts like SfiI, as might be expected from its recognition sequence, or like EcoRV, as might be expected from its crystal structure. BglI endonuclease, purified to homogeneity (41.Newman M. Lunnen K. Wilson G. Greci J. Schildkraut I. Phillips S.E.V. EMBO J. 1998; 17: 5466-5476Crossref PubMed Scopus (134) Google Scholar) by I. Schildkraut and colleagues (New England Biolabs, Beverly, MA), was a gift from M. Newman (Imperial Cancer Research Fund, London, UK). Its concentration was determined by the method of Bradford (43.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar) and is given in terms of molarity of the dimeric protein ofM r 70,000. All other enzymes were purchased from New England Biolabs; concentrations of the latter are given in terms of units of enzyme activity, as defined by the supplier. Plasmids pUC19 (44.Yanisch-Perron C. Viera C. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar), pBR322 (45.Bolivar F. Rodriguez R.L. Greene P.J. Betlach M.C. Heyneker H.L. Boyer H.W. Crosa J.H. Falkow S. Gene (Amst.). 1977; 2: 95-113Crossref PubMed Scopus (3527) Google Scholar), pAT153 (46.Twigg A.J. Sherratt D.J. Nature. 1980; 283: 216-218Crossref PubMed Scopus (650) Google Scholar), and pNEB193 (New England Biolabs) were manipulated by standard procedures (47.Sambrook J.C. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To make pBGL1 (Fig. 1 a), pUC19 was cleaved withEcoRI and KasI, and the large fragment was purified by electrophoresis prior to the removal of its single strand extensions with mung bean nuclease and re-circularization with T4 DNA ligase. To make pML1 (Fig. 1 b), pBR322 was cleaved withEcoRI and HindIII and ligated to a 43-bp duplex that had single strand extensions that matched an EcoRI terminus at one end and a HindIII terminus at the other end; the duplex was made from two self-complementary oligodeoxynucleotides, both 47 bases long, with recognition sites for Tth111I,PshAI, and AhdI that each had the same flanking and intervening sequences as the native site in pBR322. To make pAB2 (Fig. 1 c), pNEB193 was cleaved with PstI andHindIII and ligated to a 25-bp duplex with 4-base single strand extensions that matched a PstI terminus at one end and a HindIII terminus at the other end; the duplex was made from two self-complementary oligonucleotides, of 25 and 33 bases, with recognition sequences for PflMI, BstXI, andXcmI. To make pAB3 (Fig. 1 c), the small DNA fragment obtained by cleaving pAB2 with PvuII was cloned at the SspI site on pAB2; the two copies of thePvuII fragment present in pAB3 are in inverted orientation. The plasmids were used to transform recA strains ofEscherichia coli, either XL-blue or HB101 (47.Sambrook J.C. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The transformants were cultured in M9 minimal medium with 1 mCi/l [methyl-3H]thymidine and the covalently closed form of the plasmid purified by density gradient centrifugations (48.Halford S.E. Goodall A.J. Biochemistry. 1988; 27: 1771-1777Crossref PubMed Scopus (96) Google Scholar). The preparations were largely supercoiled monomeric plasmid, with <10% as either dimeric plasmid or nicked open circle DNA. Restriction enzymes were assayed by adding an aliquot (typically 5 μl) to a 3H-labeled plasmid (5 or 10 nm) in 200 μl of the buffer recommended by the supplier of the enzyme (or modifications thereof). For BglI, the aliquots were samples diluted to the requisite concentration in 10 mm Tris·HCl (pH 7.4), 300 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 500 μg/ml bovine serum albumin, and 50% (v/v) glycerol. For the other enzymes, 1–15 units of the purchased stocks were added directly to the reactions. At various times after adding the enzyme, 15-μl samples were removed from the reaction and mixed immediately with 10 μl of stop mix (21.Taylor J.D. Halford S.E. Biochemistry. 1989; 28: 6198-6207Crossref PubMed Scopus (119) Google Scholar). The samples were analyzed by electrophoresis through agarose to separate the supercoiled substrate and each of the reaction products. The segments of agarose that encompassed the substrate and each product were dissolved in 5 m sodium perchlorate and analyzed individually by scintillation counting to yield the concentration of each form of the DNA at each time point (48.Halford S.E. Goodall A.J. Biochemistry. 1988; 27: 1771-1777Crossref PubMed Scopus (96) Google Scholar, 49.Vipond I.B. Baldwin G.S. Halford S.E. Biochemistry. 1995; 34: 697-704Crossref PubMed Scopus (152) Google Scholar). For plasmids with two recognition sites, the two fragments arising from cleavage at both sites were counted together to obtain one value for the concentration of doubly cut DNA. Values of v 1 andv 2A, the rates for the utilization of the one-site and two-site substrates, respectively, were evaluated from the initial linear decline in the concentration of the supercoiled substrate with time, whereas v 2B, the rate for cutting the second site on the two-site substrate, was assessed relative to v 2A by the curve-fitting procedure described previously (11.Bilcock 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). A 465-bp fragment with one BglI site was obtained by cleaving pUC19 with AatII andEcoRI (Fig. 1 a). An isogenic 465-bp fragment that lacked a BglI site was obtained by anAatII/EcoRI digest of a mutated form of pUC19 where BglI site A, GCCATTCAGGC, had been changed to GCCATTCAGAC by using the Quikchange Mutagenesis kit (Stratagene) with primers CGCCATTCAGACTGCGCAACTG and its complement. The fragments were isolated by electrophoresis through agarose, purified with the Qiagen gel-purification kit, and labeled by using Klenow polymerase with [α-32P]dATP and dTTP (47.Sambrook J.C. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Varied amounts ofBglI (diluted in binding buffer) were added to the fragments to give 10-μl samples containing ∼50 pm DNA and 0–12.5 nm enzyme. The binding buffers contained the required concentration of NaCl in either EDTA buffer (50 mmTris·HCl (pH 7.5), 10 mm β-mercaptoethanol, 100 μg/ml bovine serum albumin, 0.1 mm EDTA) or Ca2+buffer (the same supplemented with 5 mm CaCl2). After 20 min at room temperature, loading buffer (5 μl) was added to each sample, and the samples were applied to 6% polyacrylamide gels (17.Taylor J.D. Badcoe I.G. Clarke A.R. Halford S.E. Biochemistry. 1991; 30: 8743-8753Crossref PubMed Scopus (145) Google Scholar). The loading buffer was the same as that for the binding reaction, with either EDTA or CaCl2, augmented in both cases with 40% glycerol and 0.01% (w/v) bromphenol blue. For samples in EDTA, the gel was prepared and run in 0.089 m Tris base, 0.089m boric acid, and 2 mm EDTA. For samples with Ca2+, the gel buffer was as above except for 5 mm CaCl2 in place of the EDTA (23.Vipond I.B. Halford S.E. Biochemistry. 1995; 34: 1113-1119Crossref PubMed Scopus (135) Google Scholar). After electrophoresis, the radioactivity on the gel was measured with a 400B PhosphorImager and analyzed by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). A comparison of the reactions of a restriction enzyme on substrates with either one or two recognition sites can distinguish the following schemes (11.Bilcock 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): independent reactions at each site, in the conventional distributive manner for a type II enzyme; processive reactions, where the enzyme translocates from one site to another without leaving the DNA; activation by a second site, as proposed for the type IIe enzymes; concerted action at two sites, as noted with SfiI. A type II enzyme will cleave a supercoiled (SC) plasmid with one site first in one strand to give the open circle (OC) form of the DNA and then in the other strand, to give the full-length linear (FLL) form (Fig. 2 a). The cutting of both strands is often faster than the dissociation of the enzyme from the DNA (9.Erskine S.G. Baldwin G.S. Halford S.E. Biochemistry. 1997; 36: 7567-7576Crossref PubMed Scopus (64) Google Scholar, 10.Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and steady-state reactions then yield none of the OC form (48.Halford S.E. Goodall A.J. Biochemistry. 1988; 27: 1771-1777Crossref PubMed Scopus (96) Google Scholar). A plasmid with two sites is cleaved by an enzyme that acts independently at each site in sequential steps, leading first to the transient formation of FLL DNA as one site is cut and then, after a lag phase, to the final products cut at both sites, L1 and L2 (Fig.2 b). If the recognition sites on the one-site and two-site substrates are equally susceptible to the enzyme, then independent action at each site results in the same steady-state rates for the utilization of the one- and two-site substrates (v 1 and v 2A, respectively) and for the conversion of the FLL form of the two-site DNA to L1 and L2 (v 2B). Ifv 2A = v 2B, the maximal amount of FLL DNA formed during the reaction on the two-site substrate will be 40% of the total DNA. On the other hand, both processive and concerted reactions yield less of the FLL DNA, whereas an enzyme employing a second site, either as an activator or for a concerted reaction, would consume the two-site substrate at a faster rate than the one-site substrate (11.Bilcock 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). To determine which scheme applies to the BglI enzyme, the kinetics of DNA cleavage were analyzed on plasmids with one or twoBglI sites, pBGL1 and pUC19, respectively (Fig.1 a). Under steady-state conditions with lower concentrations of enzyme than DNA (Fig.2), the rates increased linearly with increasing concentrations of the enzyme (data not shown), and both substrates were converted completely to the final products expected from the cleavage of all BglI sites: FLL DNA from pBGL1 and L1 and L2 from pUC19. Hence, BglI carries out multiple catalytic turnovers, as expected for a type II endonuclease (1.Roberts 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). The reaction of BglI on the one-site plasmid yielded none of the OC DNA and instead progressed directly to the FLL form cut in both strands (Fig. 2 a). BglI thus cleaves both DNA strands at a single recognition site at rates that are faster than its dissociation from the cleaved DNA. The two-site substrate was cleaved in sequential stages; first at one site to give FLL DNA and then, after a lag phase, at the other site to give L1 and L2 (Fig. 2 b). Moreover, the rate of utilization of the two-site substrate (v 2A = 6.3 ± 0.3 min−1) was similar to the one-site substrate (v 1 = 5.4 ± 0.6 min−1). The BglI endonuclease therefore cleaves each recognition site in an independent reaction, in the orthodox manner for a type II enzyme. The above experiments with the SC form of pUC19 do not reveal whichBglI site is cleaved to yield the FLL form. The twoBglI sites in pUC19, noted as A and B(Fig. 1 a), are flanked by different sequences, and they also have different sequences in the 5-bp spacer of nonspecific DNA in the middle of the site. Restriction activity is often affected by sequences flanking the recognition site (1.Roberts 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 activity of SfiI is also modulated by the sequence of the spacer (32.Wentzell L.M. Halford S.E. J. Mol. Biol. 1998; 281: 433-444Crossref PubMed Scopus (58) Google Scholar). Hence, oneBglI site in pUC19 may be more susceptible than the other. To monitor the cleavage of each site, pUC19 was cut withAlwNI, and the linearized DNA was used as a substrate forBglI (Fig. 3). The cleavage of either BglI site on this substrate gives rise to two fragments but the pair of fragments produced by cutting siteA are distinct from those from cutting B (Fig.3 a). A partial product of 1714 bp scores the fraction
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