Magnesium influences the discrimination and release of ADP by human RAD51.
DNA Repair(2006)
摘要
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, ATPγS, at approximately 0.08mM Mg2+. In contrast, >0.5mM 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 (0.08mM). Mg2+ did not appear to affect the ability of ATPγS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08–0.12mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (>0.3mM) 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.
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关键词
ATPase,bp,ssDNA,dsDNA,ϕX174,ADP,ATP,ATPγS,DTT,EDTA,HEPES,hRAD51,Mg2+,NPF,nt,RFI/RFII,TLC
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