title: Differential rates of NTP hydrolysis by the mutant [S69G]RecA protein. Evidence for a coupling of NTP turnover to DNA strand exchange.
abstract: The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence ((66)GPESSGKT(73)) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which serine 69 was replaced by a glycine residue. We have found that the [S69G]RecA mutation has a differential effect on the hydrolysis of various nucleoside triphosphates. The [S69G]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of rATP, ddATP, and dATP with turnover numbers of 10, 20, and 36 min(-1), respectively. The wild type RecA protein, in contrast, hydrolyzes each of these nucleoside triphosphates with similar turnover numbers of 20-24 min(-1). Significantly, the [S69G]RecA protein promotes strand exchange with all three nucleoside triphosphates, and the rate of strand exchange is directly proportional to the rate of hydrolysis of each of the nucleotide cofactors. These findings with the [S69G]RecA protein provide support for the existence of a mechanistic coupling between NTP hydrolysis and DNA strand exchange.
authors.affiliation: Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
authors.forename: S
authors.initials: S
authors.lastname: Nayak
authors.forename: F R
authors.initials: FR
authors.lastname: Bryant
mesh_terms: D004247:DNA / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016296:Mutagenesis; D009711:Nucleotides / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D009711:Nucleotides; D011994:Recombinant Proteins; D004247:DNA; D011938:Rec A Recombinases