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W2080454481 abstract "The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5′-ACTGGG-3′ and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain ofBfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism. The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5′-ACTGGG-3′ and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain ofBfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism. restriction-modification base pair(s) bis(p-nitrophenyl) phosphate bovine serum albumin kilobase pair(s) nucleotide(s) open reading frame linear DNA forms 1 and 2 Type IIs restriction enzymes recognize short non-palindromic DNA sequences and, in the presence of Mg2+ ions, cleave both DNA strands a short distance outside the recognition sequence (1Szybalski W. Kim S.C. Hasan N. Podhajska A.J. Gene ( Amst. ). 1991; 100: 13-26Crossref PubMed Scopus (197) Google Scholar). Currently, our knowledge of the structure and mechanisms of catalysis used by type IIs restriction enzymes is limited to the FokI restriction enzyme that recognizes asymmetric nucleotide sequence 5′-GGATG and cleaves both DNA strands 9/13 nucleotides away from the recognition sequence (2Sugisaki H. Kanazawa S. Gene ( Amst. ). 1981; 16: 73-78Crossref PubMed Scopus (87) Google Scholar). According to proteolytic cleavage and deletion analysis data (3Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar, 4Li L. Wu L.P. Clarke R. Gene ( Amst. ). 1993; 133: 79-84Crossref PubMed Scopus (30) Google Scholar), further confirmed by structural studies (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar), FokI contains two functional domains, one responsible for DNA recognition (N-terminal domain) and the other for cleavage (C-terminal domain). Interestingly, the structural architecture of theFokI cleavage domain displays a striking similarity to the monomer of BamHI (6Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1994; 368: 660-664Crossref PubMed Scopus (160) Google Scholar), demonstrating that both enzymes share similar catalytic machinery despite the fact that they interact with nucleic acids differently. Protein sequence comparisons suggest that the StsI restriction enzyme, which recognizes the same nucleotide sequence as FokI but cleaves DNA 10/14 nucleotides away, possesses a similar modular organization (7Kita K. Kotani H. Ohta H. Yanase H. Kato N. Nucleic Acids Res. 1992; 20: 618Crossref PubMed Scopus (9) Google Scholar, 8Kita K. Suisha M. Kotani H. Yanase H. Nucleic Acids Res. 1992; 20: 4167-4172Crossref PubMed Scopus (29) Google Scholar). However, we still lack evidence to indicate if other type IIs restriction enzymes share a similar structural architecture.The BfiI, isolated from Bacillus firmus S8120 strain, is a member of the type IIs restriction enzymes. The enzyme recognizes non-palindromic nucleotide sequence 5′-ACTGGG and cleaves complementary DNA strands 5 and 4 nucleotides beyond the recognition sequence (9Vitkute J. Maneliene Z. Petrusyte M. Janulaitis A. Nucleic Acids Res. 1998; 26: 3348-3349Crossref PubMed Scopus (17) Google Scholar). In order to gain an insight into the structural organization and mechanisms of DNA recognition and catalysis employed by the type IIs restriction enzymes, we focused on the structural, biochemical, and mechanistical characterization of the BfiI restriction enzyme. Here we report the cloning and sequence analysis ofBfiI R-M1 system and a biochemical characterization of the BfiI restriction endonuclease. We suggest that BfiI uses a novel catalytic domain to perform DNA cleavage that radically differs from the one employed by the archetypal FokI endonuclease and other type II restriction enzymes.DISCUSSIONThe restriction-modification system of the Bacillus firmus S8120 strain comprises two methyltransferases and a single restriction enzyme and is a typical type IIs system. Each methylase recognizes and methylates bases on the opposite strands of the recognition sequence making the modified DNA resistant to the restriction enzyme cleavage. Mg2+ ions are a necessary cofactor for DNA hydrolysis by type II and type IIs restriction enzymes (25Roberts 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). Biochemical experiments strikingly revealed that Mg2+ions are not required for the DNA cleavage by BfiI, raising the question of how catalysis is achieved.Based mostly on the structural and biochemical studies ofFokI endonuclease, the type IIs restriction enzymes are thought to comprise two modules connected by a flexible linker (3Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar, 4Li L. Wu L.P. Clarke R. Gene ( Amst. ). 1993; 133: 79-84Crossref PubMed Scopus (30) Google Scholar, 5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). In the case of FokI, the N-terminal subdomain is responsible for the DNA binding and the C-terminal for the cleavage. Structural comparisons revealed that the cleavage domain of FokI is structurally very similar to the monomer of dimeric type II restriction enzyme BamHI, suggesting a similar mechanism of catalysis (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). Thus, it is tempting to speculate that the FokI restriction enzyme evolved through the fusion of the catalytic machinery of the type II restriction enzyme to the separate DNA-binding domain, and developed a sophisticated mechanism to couple catalysis to sequence recognition.Strikingly, protein sequence analysis of BfiI restriction enzyme revealed (Fig. 2) that the N-terminal part of the protein exhibits weak similarities to an EDTA-resistant nuclease Nuc ofS. typhimurium (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar). The Nuc nuclease, encoded by the gene located on the pKM101 plasmid of S. typhimurium, randomly cuts single-stranded and double-stranded DNA in the absence of metal ions (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). The analysis of the protein sequence of Nuc unexpectedly revealed identities with proteins belonging to the phospholipase D superfamily (28Koonin E.V. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 29Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (282) Google Scholar). Subsequent biochemical studies of Nuc demonstrated that the enzyme catalyzes cleavage of the phosphodiester bonds via a two step mechanism involving covalent phosphohistidine intermediate of His-94 (30Gottlin E.B. Rudolph A.E. Zhao Y. Matthews H.R. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9202-9207Crossref PubMed Scopus (129) Google Scholar). Recently, the crystal structure of Nuc nuclease has been solved to 2.0-Å resolution, providing us with details of the active site organization (31Stuckey J.A. Dixon J.E. Nat. Struct. Biol. 1999; 6: 278-284Crossref PubMed Scopus (183) Google Scholar). The amino acid residues His-94, Lys-96, Ser-109, Asn-111, and Glu-122 were found in close of WO42− ion bound at the presumptive active site of Nuc. The position of the His-94 residue at the active site of Nuc is consistent with its predicted key role in catalysis.Sequence alignment between Nuc and BfiI indicates (Fig. 2) that all residues found at the active site of Nuc (including active site His) are conserved in BfiI restriction enzyme suggesting a similar organization of the active sites. It is interesting to note that secondary structure predictions for the N-terminal domain of BfiI were very similar to the secondary structure elements of Nuc, suggesting fold similarities (Fig. 2). Thus, it was tempting to suggest that N-terminal domain of BfiI is similar to Nuc. The similarities presented in Fig. 2, however, are below the statistically significant level and should be treated with caution. Therefore, we sought other evidence in support of the hypothesis that BfiI possess a Nuc-like catalytic domain.Unlike most nucleases, Nuc nuclease cleaves DNA in the absence of metal ions (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar, 26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). Restriction enzymes studied to date absolutely require Mg2+ ions for phosphodiester bond cleavage. Preliminary observations using phage λ DNA and quantitative studies of pUC19 cleavage by BfiI (Fig. 3) indicate that metal ions are unnecessary for the phosphodiester bond cleavage by BfiI and suggest mechanistic similarity to Nuc nuclease. The single turnover experiments with pUC19 and BfiI yielded the first-order rate constant of 0.052 s−1 that presumably corresponds to the rate of the chemical step (phosphodiester bond cleavage) and is independent of the metal ion. The value of the rate constant is more than 10-fold lower than values of the rate constants of the chemical step reported for the Mg2+-dependent restriction enzymesEcoRI (32Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), EcoRV (33Vermote C.L.M. Halford S.E. Biochemistry. 1992; 31: 6082-6089Crossref PubMed Scopus (89) Google Scholar), and MunI (19Sasnauskas G. Jeltsch A. Pingoud A. Siksnys V. Biochemistry. 1999; 38: 4028-4036Crossref PubMed Scopus (28) Google Scholar). The experiments with pUC19 cleavage under multiple turnover conditions (Fig. 3) revealed, however, that a step other than the chemical step, limits the overall reaction rate of pUC19 cleavage by BfiI. Indeed, the k cat for the cleavage of the closed supercoiled pUC19 form by BfiI in the absence of the metal ion was approximately 400-fold lower than the rate constant of the chemical step. It is possible that, under multiple turnover conditions, dissociation of the enzyme-product complex limits the overall reaction rate. Interestingly, metal ions (Mg2+, Mn2+, Ca2+) at 5–10 mm concentrations increased thek cat value approximately 10-fold. A similar effect has been reported for vaccinia virus topoisomerase (34Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). This enzyme did not require metal ions for the DNA cleavage; however, it exhibits metal dependence of product release rate.Moreover, like the Nuc enzyme, BfiI exhibited the ability to hydrolyze the artificial substrate bis-pNPP and metal ions were not required for catalysis. Control experiments revealed that typical type II restriction enzymes like MunI and Cfr10I or type IIs enzyme FokI did not catalyze hydrolysis of bis-pNPP either in the presence or absence of Mg ions. The reaction rate of the bis-pNPP cleavage by BfiI was much slower than the rate of DNA cleavage. The second order reaction rate constant (k cat/K m) for the bis-pNPP cleavage by BfiI was equal to the 4.2 ± 0.1m−1 s−1(pH 6.0, 25 °C). Noteworthy, the value (4.2m−1 s−1) of the second order rate constant for bis-pNPP cleavage byBfiI was close to thek cat/K m value (10m−1 s−1, 30 °C) reported for Nuc cleavage of bis-pNPP (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar).The highest rate of bis-pNPP hydrolysis both by the BfiI restriction enzyme and the Nuc nuclease was observed at pH 5.5–6.0. The alkaline limb of pH dependence of bis-pNPP hydrolysis byBfiI is consistent with the ionization of a base with an apparent pK a value of 6.4. This value is close to the pK a value of His residue and supports the assumption that such a residue is located at the active site ofBfiI. The pH dependence of bis-pNPP hydrolysis by Nuc has not been reported; however, the coincidence of the optimal pH values for bis-pNPP hydrolysis by BfiI and Nuc suggests similar pH dependence for artificial substrate hydrolysis by Nuc. In contrast to the artificial substrate, BfiI cleaved plasmid DNA both at pH 6.0 (data not shown) and pH 8.0 (Fig. 3 B). The ability to hydrolyze DNA at pH 7.5 has also been reported for the Nuc nuclease. The differences in the pH dependence values for hydrolysis of small artificial substrates and DNA by BfiI might be attributed to the perturbation of the pK a values of active site residues in the enzyme-DNA complex. If we assume that both protonated and unprotonated BfiI forms are able to bind bis-pNPP, the pK a value, determined from the pH dependence of thek cat/K m ratio corresponds to the ionization of catalytically important residue at the active site of the free enzyme (24Fersht A. Structure and Mechanism in Protein Science. W. H. Freeman and Co., New York1999Google Scholar). The pK a value of the same residue in the enzyme-DNA complex may be shifted significantly. Indeed, such effects were reported for the barnase-catalyzed hydrolysis of RNA and dinucleotides (35Gordon-Beresford R.M. Van Belle D. Giraldo J. Wodak S.J. Proteins. 1996; 25: 180-194Crossref PubMed Scopus (11) Google Scholar). The optimum pH for RNA hydrolysis of barnase was 8.5 and exceeded that GpA transesterification by 3.5 units. Alternatively, the decrease of thek cat/K m ratio with the increase of pH in the case of bis-pNPP hydrolysis by BfiI might be explained by decreased binding (increasedK m) of the low molecular weight substrate while DNA binding might be less sensitive to the pH change.Collectively, our data indicate that BfiI exhibits most of the enzymatic properties characteristic for the Nuc nuclease. However, unlike the nonspecific Nuc nuclease, BfiI restriction enzyme cleaves phosphodiester bonds in DNA site-specifically (Fig. 3,B–D). Both the specific DNA cleavage and bis-pNPP hydrolysis proceeds at the same active site of BfiI. Oligonucleotide containing the recognition sequence of BfiI effectively inhibited hydrolysis of bis-pNPP (Fig. 6) at pH 7.0. In contrast, a nonspecific oligonucleotide lacking the recognition sequence of BfiI had only a marginal effect on the rate of bis-pNPP hydrolysis. These experiments indicate that, unlike Nuc,BfiI effectively discriminates between specific and nonspecific DNA. Since sequence comparisons reveal similarities of N-terminal part of BfiI protein to the Nuc nuclease, we propose that DNA-binding specificity of BfiI stems from the C-terminal part of the protein. It is possible that, as inFokI (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar), the nucleolytic domain of BfiI is sequestered by the DNA-binding domain. Only upon BfiI binding to its recognition sequence does the cleavage domain swing over to the DNA cleavage site and the enzyme become activated. The possible cross-talking interactions between the DNA binding and cleavage domains of BfiI obviously require further studies.CONCLUSIONSThe experimental evidence presented here indicates that, in contrast to other restriction enzymes that require metal ions for catalysis, BfiI cleaves DNA specifically in the absence of metal ions. We suggest that, like to other type IIs enzymes,BfiI is composed of two subdomains that perform separate cleavage and DNA-recognition functions. The catalytic N-terminal subdomain of BfiI is presumably similar to that of nonspecific nuclease Nuc that cleaves DNA in the absence of metal ions. The C-terminal part of the BfiI presumably performs the DNA-binding function. It is tempting to speculate that BfiI evolved by fusion of the catalytic Nuc-like domain to the DNA-binding domain. The archetypal type IIs restriction enzyme FokI, in contrast to BfiI requires Mg2+ ions for DNA cleavage, its cleavage domain is located at the C-terminal part of the protein and is similar to the monomer of BamHI. Therefore, we suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differ from the archetypal FokI by the fold of the cleavage domain and by the location of the active site and reaction mechanism. Thus, type IIs restriction enzymes probably form a structurally and mechanistically diverse class. The existence of several different evolutionary lineages of type II restriction enzymes is probable. It will be interesting to see if the Nuc-like fold has been adopted by other restriction enzymes. Type IIs restriction enzymes recognize short non-palindromic DNA sequences and, in the presence of Mg2+ ions, cleave both DNA strands a short distance outside the recognition sequence (1Szybalski W. Kim S.C. Hasan N. Podhajska A.J. Gene ( Amst. ). 1991; 100: 13-26Crossref PubMed Scopus (197) Google Scholar). Currently, our knowledge of the structure and mechanisms of catalysis used by type IIs restriction enzymes is limited to the FokI restriction enzyme that recognizes asymmetric nucleotide sequence 5′-GGATG and cleaves both DNA strands 9/13 nucleotides away from the recognition sequence (2Sugisaki H. Kanazawa S. Gene ( Amst. ). 1981; 16: 73-78Crossref PubMed Scopus (87) Google Scholar). According to proteolytic cleavage and deletion analysis data (3Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar, 4Li L. Wu L.P. Clarke R. Gene ( Amst. ). 1993; 133: 79-84Crossref PubMed Scopus (30) Google Scholar), further confirmed by structural studies (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar), FokI contains two functional domains, one responsible for DNA recognition (N-terminal domain) and the other for cleavage (C-terminal domain). Interestingly, the structural architecture of theFokI cleavage domain displays a striking similarity to the monomer of BamHI (6Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1994; 368: 660-664Crossref PubMed Scopus (160) Google Scholar), demonstrating that both enzymes share similar catalytic machinery despite the fact that they interact with nucleic acids differently. Protein sequence comparisons suggest that the StsI restriction enzyme, which recognizes the same nucleotide sequence as FokI but cleaves DNA 10/14 nucleotides away, possesses a similar modular organization (7Kita K. Kotani H. Ohta H. Yanase H. Kato N. Nucleic Acids Res. 1992; 20: 618Crossref PubMed Scopus (9) Google Scholar, 8Kita K. Suisha M. Kotani H. Yanase H. Nucleic Acids Res. 1992; 20: 4167-4172Crossref PubMed Scopus (29) Google Scholar). However, we still lack evidence to indicate if other type IIs restriction enzymes share a similar structural architecture. The BfiI, isolated from Bacillus firmus S8120 strain, is a member of the type IIs restriction enzymes. The enzyme recognizes non-palindromic nucleotide sequence 5′-ACTGGG and cleaves complementary DNA strands 5 and 4 nucleotides beyond the recognition sequence (9Vitkute J. Maneliene Z. Petrusyte M. Janulaitis A. Nucleic Acids Res. 1998; 26: 3348-3349Crossref PubMed Scopus (17) Google Scholar). In order to gain an insight into the structural organization and mechanisms of DNA recognition and catalysis employed by the type IIs restriction enzymes, we focused on the structural, biochemical, and mechanistical characterization of the BfiI restriction enzyme. Here we report the cloning and sequence analysis ofBfiI R-M1 system and a biochemical characterization of the BfiI restriction endonuclease. We suggest that BfiI uses a novel catalytic domain to perform DNA cleavage that radically differs from the one employed by the archetypal FokI endonuclease and other type II restriction enzymes. DISCUSSIONThe restriction-modification system of the Bacillus firmus S8120 strain comprises two methyltransferases and a single restriction enzyme and is a typical type IIs system. Each methylase recognizes and methylates bases on the opposite strands of the recognition sequence making the modified DNA resistant to the restriction enzyme cleavage. Mg2+ ions are a necessary cofactor for DNA hydrolysis by type II and type IIs restriction enzymes (25Roberts 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). Biochemical experiments strikingly revealed that Mg2+ions are not required for the DNA cleavage by BfiI, raising the question of how catalysis is achieved.Based mostly on the structural and biochemical studies ofFokI endonuclease, the type IIs restriction enzymes are thought to comprise two modules connected by a flexible linker (3Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar, 4Li L. Wu L.P. Clarke R. Gene ( Amst. ). 1993; 133: 79-84Crossref PubMed Scopus (30) Google Scholar, 5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). In the case of FokI, the N-terminal subdomain is responsible for the DNA binding and the C-terminal for the cleavage. Structural comparisons revealed that the cleavage domain of FokI is structurally very similar to the monomer of dimeric type II restriction enzyme BamHI, suggesting a similar mechanism of catalysis (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). Thus, it is tempting to speculate that the FokI restriction enzyme evolved through the fusion of the catalytic machinery of the type II restriction enzyme to the separate DNA-binding domain, and developed a sophisticated mechanism to couple catalysis to sequence recognition.Strikingly, protein sequence analysis of BfiI restriction enzyme revealed (Fig. 2) that the N-terminal part of the protein exhibits weak similarities to an EDTA-resistant nuclease Nuc ofS. typhimurium (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar). The Nuc nuclease, encoded by the gene located on the pKM101 plasmid of S. typhimurium, randomly cuts single-stranded and double-stranded DNA in the absence of metal ions (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). The analysis of the protein sequence of Nuc unexpectedly revealed identities with proteins belonging to the phospholipase D superfamily (28Koonin E.V. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 29Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (282) Google Scholar). Subsequent biochemical studies of Nuc demonstrated that the enzyme catalyzes cleavage of the phosphodiester bonds via a two step mechanism involving covalent phosphohistidine intermediate of His-94 (30Gottlin E.B. Rudolph A.E. Zhao Y. Matthews H.R. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9202-9207Crossref PubMed Scopus (129) Google Scholar). Recently, the crystal structure of Nuc nuclease has been solved to 2.0-Å resolution, providing us with details of the active site organization (31Stuckey J.A. Dixon J.E. Nat. Struct. Biol. 1999; 6: 278-284Crossref PubMed Scopus (183) Google Scholar). The amino acid residues His-94, Lys-96, Ser-109, Asn-111, and Glu-122 were found in close of WO42− ion bound at the presumptive active site of Nuc. The position of the His-94 residue at the active site of Nuc is consistent with its predicted key role in catalysis.Sequence alignment between Nuc and BfiI indicates (Fig. 2) that all residues found at the active site of Nuc (including active site His) are conserved in BfiI restriction enzyme suggesting a similar organization of the active sites. It is interesting to note that secondary structure predictions for the N-terminal domain of BfiI were very similar to the secondary structure elements of Nuc, suggesting fold similarities (Fig. 2). Thus, it was tempting to suggest that N-terminal domain of BfiI is similar to Nuc. The similarities presented in Fig. 2, however, are below the statistically significant level and should be treated with caution. Therefore, we sought other evidence in support of the hypothesis that BfiI possess a Nuc-like catalytic domain.Unlike most nucleases, Nuc nuclease cleaves DNA in the absence of metal ions (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar, 26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). Restriction enzymes studied to date absolutely require Mg2+ ions for phosphodiester bond cleavage. Preliminary observations using phage λ DNA and quantitative studies of pUC19 cleavage by BfiI (Fig. 3) indicate that metal ions are unnecessary for the phosphodiester bond cleavage by BfiI and suggest mechanistic similarity to Nuc nuclease. The single turnover experiments with pUC19 and BfiI yielded the first-order rate constant of 0.052 s−1 that presumably corresponds to the rate of the chemical step (phosphodiester bond cleavage) and is independent of the metal ion. The value of the rate constant is more than 10-fold lower than values of the rate constants of the chemical step reported for the Mg2+-dependent restriction enzymesEcoRI (32Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), EcoRV (33Vermote C.L.M. Halford S.E. Biochemistry. 1992; 31: 6082-6089Crossref PubMed Scopus (89) Google Scholar), and MunI (19Sasnauskas G. Jeltsch A. Pingoud A. Siksnys V. Biochemistry. 1999; 38: 4028-4036Crossref PubMed Scopus (28) Google Scholar). The experiments with pUC19 cleavage under multiple turnover conditions (Fig. 3) revealed, however, that a step other than the chemical step, limits the overall reaction rate of pUC19 cleavage by BfiI. Indeed, the k cat for the cleavage of the closed supercoiled pUC19 form by BfiI in the absence of the metal ion was approximately 400-fold lower than the rate constant of the chemical step. It is possible that, under multiple turnover conditions, dissociation of the enzyme-product complex limits the overall reaction rate. Interestingly, metal ions (Mg2+, Mn2+, Ca2+) at 5–10 mm concentrations increased thek cat value approximately 10-fold. A similar effect has been reported for vaccinia virus topoisomerase (34Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). This enzyme did not require metal ions for the DNA cleavage; however, it exhibits metal dependence of product release rate.Moreover, like the Nuc enzyme, BfiI exhibited the ability to hydrolyze the artificial substrate bis-pNPP and metal ions were not required for catalysis. Control experiments revealed that typical type II restriction enzymes like MunI and Cfr10I or type IIs enzyme FokI did not catalyze hydrolysis of bis-pNPP either in the presence or absence of Mg ions. The reaction rate of the bis-pNPP cleavage by BfiI was much slower than the rate of DNA cleavage. The second order reaction rate constant (k cat/K m) for the bis-pNPP cleavage by BfiI was equal to the 4.2 ± 0.1m−1 s−1(pH 6.0, 25 °C). Noteworthy, the value (4.2m−1 s−1) of the second order rate constant for bis-pNPP cleavage byBfiI was close to thek cat/K m value (10m−1 s−1, 30 °C) reported for Nuc cleavage of bis-pNPP (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar).The highest rate of bis-pNPP hydrolysis both by the BfiI restriction enzyme and the Nuc nuclease was observed at pH 5.5–6.0. The alkaline limb of pH dependence of bis-pNPP hydrolysis byBfiI is consistent with the ionization of a base with an apparent pK a value of 6.4. This value is close to the pK a value of His residue and supports the assumption that such a residue is located at the active site ofBfiI. The pH dependence of bis-pNPP hydrolysis by Nuc has not been reported; however, the coincidence of the optimal pH values for bis-pNPP hydrolysis by BfiI and Nuc suggests similar pH dependence for artificial substrate hydrolysis by Nuc. In contrast to the artificial substrate, BfiI cleaved plasmid DNA both at pH 6.0 (data not shown) and pH 8.0 (Fig. 3 B). The ability to hydrolyze DNA at pH 7.5 has also been reported for the Nuc nuclease. The differences in the pH dependence values for hydrolysis of small artificial substrates and DNA by BfiI might be attributed to the perturbation of the pK a values of active site residues in the enzyme-DNA complex. If we assume that both protonated and unprotonated BfiI forms are able to bind bis-pNPP, the pK a value, determined from the pH dependence of thek cat/K m ratio corresponds to the ionization of catalytically important residue at the active site of the free enzyme (24Fersht A. Structure and Mechanism in Protein Science. W. H. Freeman and Co., New York1999Google Scholar). The pK a value of the same residue in the enzyme-DNA complex may be shifted significantly. Indeed, such effects were reported for the barnase-catalyzed hydrolysis of RNA and dinucleotides (35Gordon-Beresford R.M. Van Belle D. Giraldo J. Wodak S.J. Proteins. 1996; 25: 180-194Crossref PubMed Scopus (11) Google Scholar). The optimum pH for RNA hydrolysis of barnase was 8.5 and exceeded that GpA transesterification by 3.5 units. Alternatively, the decrease of thek cat/K m ratio with the increase of pH in the case of bis-pNPP hydrolysis by BfiI might be explained by decreased binding (increasedK m) of the low molecular weight substrate while DNA binding might be less sensitive to the pH change.Collectively, our data indicate that BfiI exhibits most of the enzymatic properties characteristic for the Nuc nuclease. However, unlike the nonspecific Nuc nuclease, BfiI restriction enzyme cleaves phosphodiester bonds in DNA site-specifically (Fig. 3,B–D). Both the specific DNA cleavage and bis-pNPP hydrolysis proceeds at the same active site of BfiI. Oligonucleotide containing the recognition sequence of BfiI effectively inhibited hydrolysis of bis-pNPP (Fig. 6) at pH 7.0. In contrast, a nonspecific oligonucleotide lacking the recognition sequence of BfiI had only a marginal effect on the rate of bis-pNPP hydrolysis. These experiments indicate that, unlike Nuc,BfiI effectively discriminates between specific and nonspecific DNA. Since sequence comparisons reveal similarities of N-terminal part of BfiI protein to the Nuc nuclease, we propose that DNA-binding specificity of BfiI stems from the C-terminal part of the protein. It is possible that, as inFokI (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar), the nucleolytic domain of BfiI is sequestered by the DNA-binding domain. Only upon BfiI binding to its recognition sequence does the cleavage domain swing over to the DNA cleavage site and the enzyme become activated. The possible cross-talking interactions between the DNA binding and cleavage domains of BfiI obviously require further studies. The restriction-modification system of the Bacillus firmus S8120 strain comprises two methyltransferases and a single restriction enzyme and is a typical type IIs system. Each methylase recognizes and methylates bases on the opposite strands of the recognition sequence making the modified DNA resistant to the restriction enzyme cleavage. Mg2+ ions are a necessary cofactor for DNA hydrolysis by type II and type IIs restriction enzymes (25Roberts 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). Biochemical experiments strikingly revealed that Mg2+ions are not required for the DNA cleavage by BfiI, raising the question of how catalysis is achieved. Based mostly on the structural and biochemical studies ofFokI endonuclease, the type IIs restriction enzymes are thought to comprise two modules connected by a flexible linker (3Li L. Wu L.P. Chandrasegaran S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4275-4279Crossref PubMed Scopus (166) Google Scholar, 4Li L. Wu L.P. Clarke R. Gene ( Amst. ). 1993; 133: 79-84Crossref PubMed Scopus (30) Google Scholar, 5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). In the case of FokI, the N-terminal subdomain is responsible for the DNA binding and the C-terminal for the cleavage. Structural comparisons revealed that the cleavage domain of FokI is structurally very similar to the monomer of dimeric type II restriction enzyme BamHI, suggesting a similar mechanism of catalysis (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar). Thus, it is tempting to speculate that the FokI restriction enzyme evolved through the fusion of the catalytic machinery of the type II restriction enzyme to the separate DNA-binding domain, and developed a sophisticated mechanism to couple catalysis to sequence recognition. Strikingly, protein sequence analysis of BfiI restriction enzyme revealed (Fig. 2) that the N-terminal part of the protein exhibits weak similarities to an EDTA-resistant nuclease Nuc ofS. typhimurium (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar). The Nuc nuclease, encoded by the gene located on the pKM101 plasmid of S. typhimurium, randomly cuts single-stranded and double-stranded DNA in the absence of metal ions (26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). The analysis of the protein sequence of Nuc unexpectedly revealed identities with proteins belonging to the phospholipase D superfamily (28Koonin E.V. Trends Biochem. Sci. 1996; 21: 242-243Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 29Ponting C.P. Kerr I.D. Protein Sci. 1996; 5: 914-922Crossref PubMed Scopus (282) Google Scholar). Subsequent biochemical studies of Nuc demonstrated that the enzyme catalyzes cleavage of the phosphodiester bonds via a two step mechanism involving covalent phosphohistidine intermediate of His-94 (30Gottlin E.B. Rudolph A.E. Zhao Y. Matthews H.R. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9202-9207Crossref PubMed Scopus (129) Google Scholar). Recently, the crystal structure of Nuc nuclease has been solved to 2.0-Å resolution, providing us with details of the active site organization (31Stuckey J.A. Dixon J.E. Nat. Struct. Biol. 1999; 6: 278-284Crossref PubMed Scopus (183) Google Scholar). The amino acid residues His-94, Lys-96, Ser-109, Asn-111, and Glu-122 were found in close of WO42− ion bound at the presumptive active site of Nuc. The position of the His-94 residue at the active site of Nuc is consistent with its predicted key role in catalysis. Sequence alignment between Nuc and BfiI indicates (Fig. 2) that all residues found at the active site of Nuc (including active site His) are conserved in BfiI restriction enzyme suggesting a similar organization of the active sites. It is interesting to note that secondary structure predictions for the N-terminal domain of BfiI were very similar to the secondary structure elements of Nuc, suggesting fold similarities (Fig. 2). Thus, it was tempting to suggest that N-terminal domain of BfiI is similar to Nuc. The similarities presented in Fig. 2, however, are below the statistically significant level and should be treated with caution. Therefore, we sought other evidence in support of the hypothesis that BfiI possess a Nuc-like catalytic domain. Unlike most nucleases, Nuc nuclease cleaves DNA in the absence of metal ions (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar, 26Pohlman R.F. Liu F. Wang L. More M.I. Winans S.C. Nucleic Acids Res. 1993; 21: 4867-4872Crossref PubMed Scopus (49) Google Scholar, 27Winans S.C. Walker G.C. J. Bacteriol. 1983; 154: 1117-1125Crossref PubMed Google Scholar). Restriction enzymes studied to date absolutely require Mg2+ ions for phosphodiester bond cleavage. Preliminary observations using phage λ DNA and quantitative studies of pUC19 cleavage by BfiI (Fig. 3) indicate that metal ions are unnecessary for the phosphodiester bond cleavage by BfiI and suggest mechanistic similarity to Nuc nuclease. The single turnover experiments with pUC19 and BfiI yielded the first-order rate constant of 0.052 s−1 that presumably corresponds to the rate of the chemical step (phosphodiester bond cleavage) and is independent of the metal ion. The value of the rate constant is more than 10-fold lower than values of the rate constants of the chemical step reported for the Mg2+-dependent restriction enzymesEcoRI (32Wright D.J. Jack W.E. Modrich P. J. Biol. Chem. 1999; 274: 31896-31902Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), EcoRV (33Vermote C.L.M. Halford S.E. Biochemistry. 1992; 31: 6082-6089Crossref PubMed Scopus (89) Google Scholar), and MunI (19Sasnauskas G. Jeltsch A. Pingoud A. Siksnys V. Biochemistry. 1999; 38: 4028-4036Crossref PubMed Scopus (28) Google Scholar). The experiments with pUC19 cleavage under multiple turnover conditions (Fig. 3) revealed, however, that a step other than the chemical step, limits the overall reaction rate of pUC19 cleavage by BfiI. Indeed, the k cat for the cleavage of the closed supercoiled pUC19 form by BfiI in the absence of the metal ion was approximately 400-fold lower than the rate constant of the chemical step. It is possible that, under multiple turnover conditions, dissociation of the enzyme-product complex limits the overall reaction rate. Interestingly, metal ions (Mg2+, Mn2+, Ca2+) at 5–10 mm concentrations increased thek cat value approximately 10-fold. A similar effect has been reported for vaccinia virus topoisomerase (34Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). This enzyme did not require metal ions for the DNA cleavage; however, it exhibits metal dependence of product release rate. Moreover, like the Nuc enzyme, BfiI exhibited the ability to hydrolyze the artificial substrate bis-pNPP and metal ions were not required for catalysis. Control experiments revealed that typical type II restriction enzymes like MunI and Cfr10I or type IIs enzyme FokI did not catalyze hydrolysis of bis-pNPP either in the presence or absence of Mg ions. The reaction rate of the bis-pNPP cleavage by BfiI was much slower than the rate of DNA cleavage. The second order reaction rate constant (k cat/K m) for the bis-pNPP cleavage by BfiI was equal to the 4.2 ± 0.1m−1 s−1(pH 6.0, 25 °C). Noteworthy, the value (4.2m−1 s−1) of the second order rate constant for bis-pNPP cleavage byBfiI was close to thek cat/K m value (10m−1 s−1, 30 °C) reported for Nuc cleavage of bis-pNPP (23Zhao Y. Stuckey J.A. Lohse D.L. Dixon J.E. Protein Sci. 1997; 6: 2655-2658Crossref PubMed Scopus (40) Google Scholar). The highest rate of bis-pNPP hydrolysis both by the BfiI restriction enzyme and the Nuc nuclease was observed at pH 5.5–6.0. The alkaline limb of pH dependence of bis-pNPP hydrolysis byBfiI is consistent with the ionization of a base with an apparent pK a value of 6.4. This value is close to the pK a value of His residue and supports the assumption that such a residue is located at the active site ofBfiI. The pH dependence of bis-pNPP hydrolysis by Nuc has not been reported; however, the coincidence of the optimal pH values for bis-pNPP hydrolysis by BfiI and Nuc suggests similar pH dependence for artificial substrate hydrolysis by Nuc. In contrast to the artificial substrate, BfiI cleaved plasmid DNA both at pH 6.0 (data not shown) and pH 8.0 (Fig. 3 B). The ability to hydrolyze DNA at pH 7.5 has also been reported for the Nuc nuclease. The differences in the pH dependence values for hydrolysis of small artificial substrates and DNA by BfiI might be attributed to the perturbation of the pK a values of active site residues in the enzyme-DNA complex. If we assume that both protonated and unprotonated BfiI forms are able to bind bis-pNPP, the pK a value, determined from the pH dependence of thek cat/K m ratio corresponds to the ionization of catalytically important residue at the active site of the free enzyme (24Fersht A. Structure and Mechanism in Protein Science. W. H. Freeman and Co., New York1999Google Scholar). The pK a value of the same residue in the enzyme-DNA complex may be shifted significantly. Indeed, such effects were reported for the barnase-catalyzed hydrolysis of RNA and dinucleotides (35Gordon-Beresford R.M. Van Belle D. Giraldo J. Wodak S.J. Proteins. 1996; 25: 180-194Crossref PubMed Scopus (11) Google Scholar). The optimum pH for RNA hydrolysis of barnase was 8.5 and exceeded that GpA transesterification by 3.5 units. Alternatively, the decrease of thek cat/K m ratio with the increase of pH in the case of bis-pNPP hydrolysis by BfiI might be explained by decreased binding (increasedK m) of the low molecular weight substrate while DNA binding might be less sensitive to the pH change. Collectively, our data indicate that BfiI exhibits most of the enzymatic properties characteristic for the Nuc nuclease. However, unlike the nonspecific Nuc nuclease, BfiI restriction enzyme cleaves phosphodiester bonds in DNA site-specifically (Fig. 3,B–D). Both the specific DNA cleavage and bis-pNPP hydrolysis proceeds at the same active site of BfiI. Oligonucleotide containing the recognition sequence of BfiI effectively inhibited hydrolysis of bis-pNPP (Fig. 6) at pH 7.0. In contrast, a nonspecific oligonucleotide lacking the recognition sequence of BfiI had only a marginal effect on the rate of bis-pNPP hydrolysis. These experiments indicate that, unlike Nuc,BfiI effectively discriminates between specific and nonspecific DNA. Since sequence comparisons reveal similarities of N-terminal part of BfiI protein to the Nuc nuclease, we propose that DNA-binding specificity of BfiI stems from the C-terminal part of the protein. It is possible that, as inFokI (5Wah D.A. Hirsch J.A. Dorner L.F. Schildkraut I. Aggarwal A.K. Nature. 1997; 388: 97-100Crossref PubMed Scopus (213) Google Scholar), the nucleolytic domain of BfiI is sequestered by the DNA-binding domain. Only upon BfiI binding to its recognition sequence does the cleavage domain swing over to the DNA cleavage site and the enzyme become activated. The possible cross-talking interactions between the DNA binding and cleavage domains of BfiI obviously require further studies. CONCLUSIONSThe experimental evidence presented here indicates that, in contrast to other restriction enzymes that require metal ions for catalysis, BfiI cleaves DNA specifically in the absence of metal ions. We suggest that, like to other type IIs enzymes,BfiI is composed of two subdomains that perform separate cleavage and DNA-recognition functions. The catalytic N-terminal subdomain of BfiI is presumably similar to that of nonspecific nuclease Nuc that cleaves DNA in the absence of metal ions. The C-terminal part of the BfiI presumably performs the DNA-binding function. It is tempting to speculate that BfiI evolved by fusion of the catalytic Nuc-like domain to the DNA-binding domain. The archetypal type IIs restriction enzyme FokI, in contrast to BfiI requires Mg2+ ions for DNA cleavage, its cleavage domain is located at the C-terminal part of the protein and is similar to the monomer of BamHI. Therefore, we suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differ from the archetypal FokI by the fold of the cleavage domain and by the location of the active site and reaction mechanism. Thus, type IIs restriction enzymes probably form a structurally and mechanistically diverse class. The existence of several different evolutionary lineages of type II restriction enzymes is probable. It will be interesting to see if the Nuc-like fold has been adopted by other restriction enzymes. The experimental evidence presented here indicates that, in contrast to other restriction enzymes that require metal ions for catalysis, BfiI cleaves DNA specifically in the absence of metal ions. We suggest that, like to other type IIs enzymes,BfiI is composed of two subdomains that perform separate cleavage and DNA-recognition functions. The catalytic N-terminal subdomain of BfiI is presumably similar to that of nonspecific nuclease Nuc that cleaves DNA in the absence of metal ions. The C-terminal part of the BfiI presumably performs the DNA-binding function. It is tempting to speculate that BfiI evolved by fusion of the catalytic Nuc-like domain to the DNA-binding domain. The archetypal type IIs restriction enzyme FokI, in contrast to BfiI requires Mg2+ ions for DNA cleavage, its cleavage domain is located at the C-terminal part of the protein and is similar to the monomer of BamHI. Therefore, we suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differ from the archetypal FokI by the fold of the cleavage domain and by the location of the active site and reaction mechanism. Thus, type IIs restriction enzymes probably form a structurally and mechanistically diverse class. The existence of several different evolutionary lineages of type II restriction enzymes is probable. It will be interesting to see if the Nuc-like fold has been adopted by other restriction enzymes." @default.
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W2080454481 title "Novel Subtype of Type IIs Restriction Enzymes" @default.
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