title: A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site.
abstract: Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.
authors.affiliation: Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Sasnauskas
authors.forename: Linas
authors.initials: L
authors.lastname: Zakrys
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaremba
authors.forename: Richard
authors.initials: R
authors.lastname: Cosstick
authors.forename: James W
authors.initials: JW
authors.lastname: Gaynor
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D010710:Phosphates / Q000378:metabolism
chemical_list: C465503:3'-S-phosphorothiolate; D010710:Phosphates; D006639:Histidine; D004247:DNA; C113635:endodeoxyribonuclease BfiI; D015252:Deoxyribonucleases, Type II Site-Specific