Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Abstract

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5′-GAATGCN↓-3′/5′-NG↓CATTC-3′ and cuts top and bottom DNA strands at positions, indicated by the “↓” symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two “PD-(D/E)XK” domains. The N-terminal domain is related to the 5′-GAATTC-3′-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites. Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5′-GAATGCN↓-3′/5′-NG↓CATTC-3′ and cuts top and bottom DNA strands at positions, indicated by the “↓” symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two “PD-(D/E)XK” domains. The N-terminal domain is related to the 5′-GAATTC-3′-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites. Type II restriction enzymes (REases) 5The abbreviations used are: REaserestriction endonucleasewtwild-typeFRfoldrecognitionBSAbovine serum albuminaaamino acid(s)SCsupercoiledOCopen-circle.5The abbreviations used are: REaserestriction endonucleasewtwild-typeFRfoldrecognitionBSAbovine serum albuminaaamino acid(s)SCsupercoiledOCopen-circle. are one of the largest groups of endonucleases. They recognize short double-stranded DNA sequences and specifically cleave both strands of DNA at fixed positions within or near these sites (1Roberts R.J. Belfort M. Bestor T. Bhagwat A.S. Bickle T.A. Bitinaite J. Blumenthal R.M. Degtyarev S Kh., Dryden D.T.F. Dybvig K. Firman K. Gromova E.S. Gumport R.I. Halford S.E. Hattman S. Heitman J. Hornby D.P. Janulaitis A. Jeltsch A. Josephsen J. Kiss A. Klaenhammer T.R. Kobayashi I. Kong H. Kruger D.H. Lacks S. Marinus M.G. Miyahara M. Morgan R.D. Murray N.E. Nagaraja V. Piekarowicz A. Pingoud A. Raleigh E. Rao D.N. Reich N. Repin V.E. Selker E.U. Shaw P.C. Stein D.C. Stoddard B.L. Szybalski W. Trautner T.A. Van Etten J.L. Vitor J.M. Wilson G.G. Xu S.Y. Nucleic Acids Res. 2003; 31: 1805-1812Crossref PubMed Scopus (539) Google Scholar), a property that has made them indispensable for DNA engineering. All structurally characterized REases have been found to share a common three-dimensional fold harboring a bipartite PD-Xn-(D/E)XK pattern of three moderately conserved charged catalytic/Mg2+ binding residues (2Bujnicki J.M. Curr. Protein Pept. Sci. 2003; 4: 327-337Crossref PubMed Scopus (54) Google Scholar, 3Pingoud A. Fuxreiter M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (374) Google Scholar). However, their amino acid sequences are strongly divergent. Typically, only enzymes that recognize identical or very similar DNA sequences may display similarity at the amino acid level (4Pingoud V. Sudina A. Geyer H. Bujnicki J.M. Lurz R. Luder G. Morgan R. Kubareva E. Pingoud A. J. Biol. Chem. 2005; 280: 4289-4298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Recent bioinformatics analyses suggested that REases belong also to at least three other folds, GIY-YIG, HNH, and PLD (5Aravind L. Makarova K.S. Koonin E.V. Nucleic Acids Res. 2000; PubMed Google Scholar, J.M. M. L. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. A. G. A. V. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the catalytic and DNA residues of REases is restriction endonuclease serum amino restriction endonuclease serum amino Most of II REases recognize DNA sequences and cleave both DNA strands within the target at They as in which of two identical with the of the DNA sequence A. Fuxreiter M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (374) Google Scholar). Type II REases that recognize asymmetric are as R.J. T. J. Nucleic Acids Res. 2003; 31: PubMed Scopus Google Scholar). are that cleave both DNA strands within the sequence. of and have been characterized Wilson G.G. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, K. A. A. Janulaitis A. Nucleic Acids Res. PubMed Scopus Google Scholar). enzymes are of two but that and recognize and cleave DNA in an of Type REases are enzymes that cleave at least one strand of the DNA outside of the sequence (1Roberts R.J. Belfort M. Bestor T. Bhagwat A.S. Bickle T.A. Bitinaite J. Blumenthal R.M. Degtyarev S Kh., Dryden D.T.F. Dybvig K. Firman K. Gromova E.S. Gumport R.I. Halford S.E. Hattman S. Heitman J. Hornby D.P. Janulaitis A. Jeltsch A. Josephsen J. Kiss A. Klaenhammer T.R. Kobayashi I. Kong H. Kruger D.H. Lacks S. Marinus M.G. Miyahara M. Morgan R.D. Murray N.E. Nagaraja V. Piekarowicz A. Pingoud A. Raleigh E. Rao D.N. Reich N. Repin V.E. Selker E.U. Shaw P.C. Stein D.C. Stoddard B.L. Szybalski W. Trautner T.A. Van Etten J.L. Vitor J.M. Wilson G.G. Xu S.Y. Nucleic Acids Res. 2003; 31: 1805-1812Crossref PubMed Scopus (539) Google Scholar). of enzymes the the sequence and the site of by an enzymes that cleave both DNA strands the target comprises two and one for DNA binding and for which are by a I. PubMed Scopus Google Scholar). to DNA as a monomer the DNA binding the of the catalytic of two and cleave the DNA at a fixed near one of the sites J. I. Sci. S. A. PubMed Scopus Google Scholar, E.S. S. J. Mol. Biol. PubMed Scopus Google Scholar, Bitinaite J. I. Sci. S. A. PubMed Scopus Google Scholar). dimerization is for double-stranded DNA mutants in dimerization DNA cleavage J. I. Sci. S. A. PubMed Scopus Google Scholar). is that other REases of in a similar S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Morgan R.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), of them cleave one strand of cognate DNA. dimerization is only to the cleavage of the DNA strand Kong H. Nucleic Acids Res. PubMed Google Scholar, Kong H. Sci. S. A. PubMed Scopus Google Scholar). The other of REases that cleave both DNA strands very to the to by the of the DNA target sequence and the the cleavage that the FokI-like DNA and cleavage is to these by S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and at nicking enzymes of of enzymes Xu S.Y. Nucleic Acids Res. PubMed Scopus (29) Google Scholar, J. A. Xu S.Y. J. Mol. Biol. PubMed Scopus Google suggested that REases may two active sites. However, in The is a that recognizes an asymmetric DNA sequence 5′-GAATGCN↓-3′/5′-NG↓CATTC-3′ and cuts top and bottom DNA strands at positions indicated by V. L. J. and V. V. L. J. and V. have that are two the DNA cleavage and of Mva1269I bioinformatics and Our that Mva1269I is a enzyme two active sites for the cleavage of DNA strand, which has evolved by fusion of a nuclease domain, similar to EcoRI, to a nonspecific nuclease domain, similar to FokI. and the used as a in and used for of All in at to J. T. Scholar). of E. by the or by J. T. Scholar). All molecular and the Mva1269I DNA with and DNA by DNA and The with two Mva1269I as The DNA of with and the with a DNA identical in sequence to the site of and the Mva1269I target in the The by The used in DNA cleavage by in DNA restriction of and of DNA J. T. Scholar). The sequence the DNA and DNA and as J. T. Scholar). the of the M. of the for Mva1269I and of the for Mva1269I are upon and of Mva1269I and the DNA of in Mva1269I the for Mva1269I into J. PubMed Google Scholar). The DNA to Mva1269I the for Mva1269I into the of the and into of Mva1269I by the G. Google Scholar). The of the as well as the of by DNA the of are upon Mva1269I and its to and with at the by and in The and by The to and with Mva1269I and at Mva1269I mutants the double-stranded cleavage the However, in of by and that the to that of the enzyme The of by with and to their of the PubMed Scopus Google Scholar). sequences for the top strand of double-stranded DNA for Mva1269I target the of and Mva1269I The sequences of the used for the bottom strand and All as a of and They used of the and cleavage top or the bottom strands of the with by that are to the the used in a of by the solution to in a and to by of DNA of and by at with DNA in and at and the cleavage by of The by and with The and DNA DNA of by Mva1269I at with DNA in and Mva1269I at the by of The by DNA of by with and or of the and Mva1269I in at at and the cleavage by of solution for at and in The by DNA and by and Life DNA binding by Mva1269I and by by the the or nonspecific the the of the of the enzyme, the for at with of the enzyme in of binding with or with and at in which with in used in the the and a with of enzymes in the of DNA the with Mva1269I in binding with and for at of an DNA a site for The by as The DNA by as a and by of DNA nicked with the Mva1269I at The nicked and used as a for The and used in or with in a of a to in and The at in an a at for the top and by The by and DNA as a The serum and of as that of with a DNA of and of or with a of DNA and the of the of a DNA and of a and and Protein and fold-recognition of similarity to sequence the at Bujnicki J.M. Nucleic Acids Res. 2003; 31: PubMed Scopus Google Scholar). the is to at the level of the analysis by the Mva1269I sequence into a of to the of amino to the of used as a for the J. M. J.M. Bujnicki J.M. 2003; PubMed Scopus Google Scholar), of in and of which of the of the the E. J. Mol. Biol. PubMed Scopus Google Scholar). has to of by the results of the at of REases and other Bujnicki J.M. Biol. 2005; PubMed Scopus Google Scholar, M. Bujnicki J.M. Protein 2005; PubMed Scopus Google Scholar, M. Bujnicki J.M. 2005; PubMed Scopus Google Scholar, M. Bujnicki J.M. Nucleic Acids Res. 2005; PubMed Scopus Google Scholar). The is to the for a of the Mva1269I of and of the M. DNA region for the Mva1269I and revealed that the Mva1269I of two which are by The for the Mva1269I of amino acid residues Mva1269I amino acid sequence with its The the of amino acid residues analysis revealed that is of two sequence similarity to DNA that A. V. Janulaitis A. PubMed Scopus Google Scholar). DNA by of DNA cleavage by an of that the enzyme with one and two sites at similar and the DNA by at site S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these the suggested that to with two target sites and that is a monomer with two active one for the cleavage of DNA strand. Mva1269I the and Mva1269I The enzyme found to DNA cleavage by the of with one site and two sites as Mva1269I the the of the of a nicked the the cleavage of both DNA strands is and Mva1269I the target the the of the of Mva1269I with the the of the DNA the by cleavage of the site the that the two cleavage are and the of DNA cleavage similar of the of Mva1269I and for and is in with the cleavage of the and of other of which require two sites for S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The DNA cleavage of Mva1269I are very similar to of and of which are S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to Mva1269I by other REases recognize DNA and double-stranded DNA Mva1269I a in of and Mva1269I by of Mva1269I in the of DNA that Mva1269I as a of The of Mva1269I is Our suggest that Mva1269I is a monomer in the of as found for other REases T. PubMed Scopus Google Scholar, M. T. Nucleic Acids Res. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Mva1269I a with DNA is well that REases require for DNA and in the of Halford S.E. PubMed Scopus Google Scholar). DNA binding revealed that Mva1269I and nonspecific DNA in the of the catalytic and that binding in the of used in Mva1269I in the of the enzyme with a or of DNA and to for Mva1269I and for their of of them the DNA to the an molecular of is The molecular of DNA by its in with that by The with the of Mva1269I that Mva1269I and DNA a with of The molecular of the the of molecular of one and two DNA However, the of and DNA revealed that only a of the DNA with Mva1269I in a and of the DNA DNA in a results suggested that a monomer of Mva1269I a with DNA. results by a the by and catalytic used of the to Mva1269I cleavage of cleavage of and with the at the cleavage and cleavage of the top and bottom The strand of is as one or two DNA with the Mva1269I a DNA The by Mva1269I with the the DNA with a Mva1269I site in to the a monomer of Mva1269I with two DNA an a and two DNA of the the new to the as a DNA. that in the of one of Mva1269I one DNA to a these results the of Mva1269I a double-stranded DNA Mva1269I has two active sites one for DNA strand, or Mva1269I has only one active which one DNA strand and in the the DNA strand. two active sites of of them Mva1269I into a nicking a enzyme only one active site to cleave both DNA for active sites and their analysis to these two of of in the of residues in used the fold-recognition to for of The N-terminal region found to related to the to the to three other and with the top to the by the The C-terminal region found to related to the catalytic domain of to and to and with the top by The region of Mva1269I similarity to The sequence by the sequence to used the J. M. J.M. Bujnicki J.M. 2003; PubMed Scopus Google Scholar). to and amino acid analysis of the and sequence revealed that the C-terminal domain a active site a as a for an catalytic in the sequence in The N-terminal domain a similar However, its catalytic is and the in the of REases that of the common catalytic residues and are R. S. R. V. J. Mol. Biol. PubMed Scopus Google Scholar). used the to and The in the C-terminal FokI-like domain of Mva1269I for of the amino acid residues and the catalytic in the C-terminal domain are in DNA cleavage by mutants and as well as a by and the to and cleave the DNA and whereas the and mutants but double-stranded DNA cleavage and a DNA of in with and their for at and suggested that a short double-stranded DNA the of the DNA and DNA the are two Mva1269I of the cleavage sites found in DNA. are the of DNA positions and the of the the to the nicking of Mva1269I by in of the DNA the which has a target at the of DNA the of into two of the and The of Mva1269I mutants both the which a Mva1269I target and its The of into the by mutants and at a the with of both mutants with that the nicking of of of the of double-stranded DNA at or to that the cleavage of the strand by the the and mutants double-stranded DNA cleavage at elevated in of of the All these results with the DNA as a is that of the enzyme to the into the that the DNA nicking of mutants and is with the double-stranded of the DNA nicking by the nicked of with a enzyme, and to The DNA the DNA strand that the bottom strand of the Mva1269I target used as a at the which with the of Mva1269I cleavage the bottom strand. DNA the top strand used as a results nicked by or and the mutants and are nicking enzymes that only the bottom strand of the Mva1269I that the an active site in the cleavage of the top strand. and to the in the N-terminal EcoRI-like of Mva1269I in of the the of the active site in the EcoRI-like domain, mutants of and by and the identical only with the are The cleavage DNA at very or of and a the of the which been for as in the nicking mutants of the C-terminal domain of the with the DNA has to its into the nicked However, a of the to of the in with the of the of the C-terminal active site for nicking a of DNA with the of the the the DNA of the with the enzyme with the to a nicking endonuclease with a double-stranded DNA cleavage We that mutants in the and C-terminal domain of Mva1269I may for nicking of strand of the the specifically the top strand target, to the in which the bottom strand is whereas the nicked by the by the of nicking We that the nicked of by into the by both the enzyme and by the and nicked by by both the enzyme and by and of nicked by that the top strand of cognate DNA The only the DNA strand the top strand of the Mva1269I target as a at the with the top strand cleavage of the Mva1269I target, whereas the bottom strand of the target that to the N-terminal active which is for the cleavage of the bottom DNA strand, and that mutants of that active site cleave the top strand of DNA However, the cleavage of the bottom strand is by or The of Mva1269I and the of the catalytic amino acid residues the of DNA binding the DNA of used as of to as a Mva1269I target in the very of the sequence. The to as three in the Mva1269I target We that the enzyme a with DNA and the nonspecific in the of that a of DNA is into a by a in Most a in which one Mva1269I is specifically to the target and one is to the DNA as with other REases A. S. E. V. PubMed Scopus Google Scholar). DNA binding of the nicking mutants and revealed that DNA at to of the enzyme a for the nonspecific DNA in the active site of the C-terminal domain have of the is in with of active site of other which the A. Fuxreiter M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (374) Google Scholar, E. J. A. A. 2005; PubMed Scopus (29) Google Scholar). However, the nicked only by a of or that of the enzyme to the of the identical of the enzyme and that the in the of the DNA nicking mutants is the of the of enzymes their nicked DNA cleavage that of the with the in the N-terminal active site to of the target with mutants of the C-terminal active site indicated that the nicking of mutants a by a for DNA. However, DNA binding that of are identical to for the enzyme that other of in the nicking Mva1269I DNA binding and cleavage revealed that mutants of the C-terminal active site are active nicking enzymes mutants of the N-terminal active a of by the DNA cleavage in which the cleavage of the bottom strand by the N-terminal active site is a prerequisite for the cleavage of the top strand by the C-terminal active of the the by at the site of cleavage in DNA strand or cleavage by REases Pingoud A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). both catalytic sites of Mva1269I cleave their DNA strands the of in one DNA strand with the cleavage of the other strand. However, the cleavage of the top strand is by the cleavage of the bottom strand, of the cleavage the cleavage of both strands to the to the of the Mva1269I with with a at the of cleavage of the top strand, with a at the of cleavage of the bottom strand, or with two that are at positions of cleavage of the top and bottom that the used in a of and which of cleavage by the of cleavage of by the of both strands with identical that cleavage of both strands is and the results of cleavage of nicked of the by at the site of cleavage in the top strand the of cleavage of strand by However, the bottom strand as as in DNA and by of cleavage in and that the cleavage of the bottom strand is the cleavage of the top strand. the in the bottom strand of DNA the cleavage of both strands that cleavage of the top strand the cleavage of the bottom DNA strand. results a of double-stranded DNA cleavage by Mva1269I and the nicking in the and C-terminal active sites. The results for cleavage of the with in both DNA that Mva1269I the bottom DNA strand by in the top strand. the other the top strand only a the of cleavage of the bottom strand one that Mva1269I cleave the bottom strand in the with a to that in the cleavage of both DNA strands that the at the of the top strand cleavage cleavage of the bottom strand. is also in with the that the bottom strand of the the in the top strand, the However, are to the of at the of cleavage in one strand the cleavage of the strand. two for double-stranded DNA cleavage have been for II REases and related one of two identical active sites are by dimerization of the catalytic domains. The DNA target is in a The may at the but in the of both nuclease with the DNA and with its are is common for the REases as EcoRI, the enzymes A. Fuxreiter M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (374) Google Scholar), and for endonucleases the and Stoddard B.L. Nucleic Acids Res. PubMed Scopus Google that within their target sites. The is used by the as by catalytic to cleave a nonspecific sequence the target the of two but nuclease in two as in the of enzymes Wilson G.G. J. Mol. Biol. 2005; PubMed Scopus Google or K. A. A. Janulaitis A. Nucleic Acids Res. PubMed Scopus Google or within a as in the of endonucleases the as S. Wende W. S. Pingoud A. Pingoud V. J. PubMed Scopus Google Scholar). the cleavage of both strands The the of a active which is used for the cleavage of both of suggested for the enzyme a to the DNA a C-terminal domain and the N-terminal nuclease domain to a double-stranded with of the enzyme M. Belfort M. J. PubMed Scopus Google Scholar). similar may by the active site is by two PLD nuclease G. Halford S.E. V. Sci. S. A. 2003; PubMed Scopus Google Scholar). that a a of the and its target as a monomer revealed by the but in a dimerization R.J. Full Text Full Text PDF PubMed Scopus Google Scholar). a of a by a which recognizes an asymmetric sequence and two nuclease to cleave both strands fold-recognition analyses that the N-terminal domain of Mva1269I is but related to EcoRI, a enzyme, which recognizes a sequence and cuts as indicated by the “↓” symbol. the other the C-terminal domain of Mva1269I to related to the nonspecific nuclease domain of the to that the pattern of and cleavage of the bottom strand by Mva1269I is similar to that of whereas the cleavage of the top strand in a nonspecific region outside the sequence to FokI. of the amino acid residues to the active sites revealed that the EcoRI-like domain is for the whereas the FokI-like domain is for the acid in active site nicking enzymes with very The DNA binding and cleavage analysis of the enzyme and the mutants revealed that the cleavage of the bottom strand the cleavage of the top strand. Mva1269I two active enzymes with two identical or related nuclease but them to cleavage of both as found with enzymes with only one active is that the pattern of cleavage by Mva1269I and the of cleavage of its and are very which that the of the two active sites in Mva1269I is to of related which have their pattern of cleavage by of the have been well in the M. Bujnicki J.M. Nucleic Acids Res. 2005; PubMed Scopus Google Scholar, M. K. Wilson G. J. I. S.E. J. PubMed Scopus Google Scholar, V. Kubareva E. G. Bujnicki J.M. Sudina A. Pingoud A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of a which has evolved a cleavage pattern by two to the two nuclease in Mva1269I are and in the with and the DNA or the is and both may that both nuclease of Mva1269I to the target sequence or by the of the in the The strongly suggest that is the N-terminal EcoRI-like domain, which the of is to the within the target which only the FokI-like domain to the by the other strand outside the sequence. both the and of Mva1269I an of domain The of the EcoRI-like and FokI-like have been but within the Mva1269I are with to other in a to sequence analyses revealed that other enzymes that cleave both DNA strands very to their sequence as and Xu S.Y. Nucleic Acids Res. PubMed Scopus (29) Google Scholar, J. A. Xu S.Y. J. Mol. Biol. PubMed Scopus Google also a of strongly active sites. The DNA cleavage of these REases have been S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). All of these the Mva1269I which amino acid sequence cleave a site or two sites with a similar They require two of their and REases S.E. Halford S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have been to these enzymes double-stranded the of two active sites and by the of and mutants to top or bottom DNA strands Xu S.Y. Nucleic Acids Res. PubMed Scopus (29) Google Scholar, J. A. Xu S.Y. J. Mol. Biol. PubMed Scopus Google Scholar). However, that the of that of the in to the DNA Xu S.Y. Nucleic Acids Res. PubMed Scopus (29) Google Scholar), whereas the of cleavage by Mva1269I is the DNA cleavage is used by other However, the similarity of Mva1269I and as well as the of amino acid residues in the of both Mva1269I active sites to that the into a nicking The of Mva1269I and other REases that enzymes of for only used to in but the by at the sequence the of the other Our results also suggest that may to REases by of the nuclease of has been that a of and to the engineering of with new T. R.J. Stoddard B.L. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). Our of Mva1269I is of to but as a for to and as a of enzyme is We are to for the Mva1269I We and for and of the for enzymes and