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 Mg 2+ compared to RecA. We have further explored the effect of Mg 2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPγS, at approximately 0.08 mM Mg 2+ . In contrast, >0.5 mM Mg 2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg 2+ concentrations (0.08 mM). Mg 2+ did not appear to affect the ability of ATPγS to competitively inhibit the hRAD51 ATPase. Low Mg 2+ (0.08–0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg 2+ concentration (>0.3 mM) was inhibitory. At low Mg 2+ , 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 Mg 2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg 2+ with EDTA significantly enhanced ADP → ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg 2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.