The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre

Bfi I is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg2+. The amino acid sequence of the N-terminal part of Bfi I has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of Bfi I that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of Bfi I were all catalytically inactive but were still able to bind DNA specifically. The active site of Bfi I is thus likely to be similar to that of Nuc. Bfi I was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of Bfi I binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, Bfi I cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the Bfi I dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site. (C) 2003 Elsevier Science Ltd. All rights reserved.