Magnesium influences the discrimination and release of ADP by human RAD51.
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.
Shim
Kang-Sup
KS
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
Tombline
Gregory
G
Heinen
Christopher D
CD
Charbonneau
Nicole
N
Schmutte
Christoph
C
Fishel
Richard
R
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
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