title: 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.
authors.affiliation: Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania.
authors.forename: Elena
authors.initials: E
authors.lastname: Armalyte
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.forename: Jolanta
authors.initials: J
authors.lastname: Giedriene
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.forename: Jan
authors.initials: J
authors.lastname: Kosiński
authors.forename: Arvydas
authors.initials: A
authors.lastname: Lubys
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D003460:Crystallization; D004247:DNA / Q000737:chemistry; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D007700:Kinetics; D008837:Micrococcus / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D009841:Oligonucleotides; D004247:DNA; D015246:Deoxyribonuclease EcoRI; C053864:endodeoxyribonuclease BsmI; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D002118:Calcium