title: Magnesium influences the discrimination and release of ADP by human RAD51.
abstract: hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.
authors.affiliation: Department of Molecular Virology, Immunology, and Medical Genetics, Human Cancer, Genetics, The Ohio State University College of Medicine, The Ohio State University, Comprehensive Cancer Center, Columbus, OH 43102, USA. shim55@osu.edu
authors.forename: Kang-Sup
authors.initials: KS
authors.lastname: Shim
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Heinen
authors.forename: Nicole
authors.initials: N
authors.lastname: Charbonneau
authors.forename: Christoph
authors.initials: C
authors.lastname: Schmutte
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
mesh_terms: D000241:Adenosine / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015374:Biosensing Techniques; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000378:metabolism*; D011938:Rec A Recombinases / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D008274:Magnesium; D000241:Adenosine