title: Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex.
abstract: The kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex were examined by stopped-flow fluorescence spectroscopy, using a modified version of the RecA protein that contains a fluorescent reporter group in the ssDNA binding site. The results indicated that: i) an active RecA-ssDNA complex was formed more rapidly on dT200 than on dT50 when either ATP or dATP was provided as the nucleotide cofactor, and ii) active complex formation occurred more rapidly with dATP than with ATP on either dT50 or dT200. The dependence on both the identity of the nucleotide cofactor and the length of the ssDNA effector indicated that active complex formation occurs by a cooperative mechanism and that dATP is more effective than ATP in mediating the interactions between RecA monomers that drive this process. Interestingly, the time courses of dATP-mediated active complex formation were closely similar to those that were obtained with ATPγS, an effectively non-hydrolyzable ATP analog that strongly stabilizes the active conformation of the RecA-ssDNA complex. These results provide mechanistic insight into the enhanced ssDNA binding and DNA strand exchange activities that are observed when dATP is provided in place of ATP in RecA biochemical assays.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Sunil
authors.initials: S
authors.lastname: Nayak
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address: fbryant1@jhu.edu.
authors.forename: Floyd R
authors.initials: FR
authors.lastname: Bryant
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D003839:Deoxyadenosines / Q000737:chemistry*; D005453:Fluorescence; D007700:Kinetics
chemical_list: D004277:DNA, Single-Stranded; D003839:Deoxyadenosines; D000255:Adenosine Triphosphate; C058118:2'-deoxyadenosine
keywords: Conformational change; Nucleotide cofactor; RecA protein; Stopped-flow fluorescence