Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition.
RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5'-O-(thiotriphosphate) (ATPgammaS). hRAD51 fully saturates with ATP/ATPgammaS regardless of DNA cofactor (K(D) approximately 5 microm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPgammaS binding (K(app1) approximately 3 microm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K(app2) > or = 125 microm; >1 ADP/1 hRAD51). We could detect ADP --> ATP exchange in the high affinity ADP binding mode (K(app1)) but not the low affinity binding mode (K(app2)). At low ATP concentrations (<0.3 mm), ADP and ATPgammaS competitively inhibit the hRAD51 ATPase (K(m)((app)) > K(m)). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPgammaS that were 20-fold above the K(D). Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially ( approximately 10-fold) and ATP modestly ( approximately 3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.
Tombline
Gregory
G
Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Shim
Kang-Sup
KS
Fishel
Richard
R
D000227:Adenine Nucleotides / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000645:Ammonium Sulfate / Q000737:chemistry; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051135:Rad51 Recombinase; D013095:Spermidine / Q000737:chemistry
D000227:Adenine Nucleotides; D004268:DNA-Binding Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; D000645:Ammonium Sulfate; D013095:Spermidine