title: Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily.
abstract: The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.
authors.affiliation: Institute of Biotechnology, Graiciuno 8, Vilnius 2028, Lithuania.
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaremba
authors.forename: Claus
authors.initials: C
authors.lastname: Urbanke
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D005810:Multigene Family; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; C113635:endodeoxyribonuclease BfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D010739:Phospholipase D