title: The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre.
abstract: BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI 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 BfiI 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 BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI 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 BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.
authors.affiliation: Institute of Biotechnology, Graiciuno 8, Vilnius 2028, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Lagunavicius
authors.forename: Giedrius
authors.initials: G
authors.lastname: Sasnauskas
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008836:Micrococcal Nuclease; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; C074002:nuc protein, staphylococcus; D004247:DNA; D004720:Endonucleases; C113635:endodeoxyribonuclease BfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D008836:Micrococcal Nuclease