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Cloning and characterization of a new site specificmethyl-directed DNAendonuclease EcoBLI recognizing 5-G(5mC)NGC-3/3- CGN(5mC)G-5

D.A.Gonchar, V.A.Chernukhin,M.A.Abdurashitov, E.V.Kileva, V.S.Dedkov, N.A.Mikhnenkova, E.N.Lomakovskaya, S.G.Udalyeva, S.Kh. Degtyarev

A gene coding BisI, site specific 5mC-directed DNA endonuclease recognizing DNA sequence 5Â’-G(5mC)NGC-3Â’/3Â’-CGN(5mC)G-5Â’, was recently identified in the sequenced genome of the strain-producer Bacillus subtilis T30. In this work we have undertaken a search of bisI gene homologues among the sequenced genomes of enterobacteria. DNA analysis has revealed a small group of highly homologous ORFs with unknown function including one ORF inDNA ofwell-known strain E.coli. This ORF WP 001276099.1 from E.coli BL21 (DE3) was amplified and cloned. An obtained recombinant strain E.coli pEcoBLI produces MDendonuclease named EcoBLI. The new enzyme has the same substrate specificity as BisI MD-endonuclease. Thus, ORFWP 001276099.1 from E.coli BL21 (DE3) encodes site-specific 5mC-directed DNAendonuclease EcoBLI recognizing and cleaving DNA sequence as indicated by arrows 5Â’-G(5mC)^NGC-3Â’/3Â’-CGN^(5mC)G-5Â’.