Novel subtype of type IIs restriction enzymes - BfiI endonuclease exhibitssimilarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleot ide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleot ides 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 sy stem contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme, Sequencing of bisulfite-treated methylated DNA indic ated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzym e 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 M g2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphat e. However, unlike the nonspecific S. typhimurium nuclease, BfiI restrictio n enzyme cleaves DNA specifically. We propose that the DNA-binding specific ity of BfiI stems from the C-terminal part of the protein. The catalytic N- terminal subdomain of BfiI radically differs from that of type II restricti on enzymes and is presumably similar to the EDTA-resistant nonspecific nucl ease of S. typhimurium; therefore, BfiI did not require metal ions for cata lysis. We suggest that BfiI represents a novel subclass of type IIs restric tion enzymes that differs from the archetypal KokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.