title: Nucleotide binding studies of bacteriophage T7 DNA helicase-primase protein.
abstract: Bacteriophage T7 DNA helicase protein is a hexameric protein that contains identical subunits arranged in a ring-like structure. Single-stranded DNA binds through the hole of the ring, and the helicase protein translocates and unwinds duplex DNA using nucleoside triphosphate (NTP) hydrolysis. In our efforts to understand how NTP hydrolysis may be coupled to movement of the helicase on the DNA, we have quantitated the equilibrium binding of deoxythymidine triphosphate and thymidine 5'-(beta,gamma-methylenetriphosphate) using nitrocellulose binding assays. Even though the helicase consists of six identical subunits, each hexamer was found to bind only three NTP molecules. These results indicate half-site binding or negative cooperativity in NTP binding by the hexamer. Interestingly, binding of three NTP molecules to the hexamer was sufficient for stoichiometric binding of a single-stranded oligodeoxynucleotide. Similar negative cooperativity in NTP binding has also been observed for other helicases, suggesting that it may be a general feature of hexameric helicases. The significance of half-site binding, however, is not understood at the present time.
authors.affiliation: Department of Biochemistry, Ohio State University, Columbus 43210, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Patel
authors.forename: M M
authors.initials: MM
authors.lastname: Hingorani
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D009711:Nucleotides; D013942:Thymine Nucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C024157:thymidine 5'-triphosphate