title: Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition.
abstract: 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.
authors.affiliation: Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.forename: Kang-Sup
authors.initials: KS
authors.lastname: Shim
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
mesh_terms: 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
chemical_list: D000227:Adenine Nucleotides; D004268:DNA-Binding Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; D000645:Ammonium Sulfate; D013095:Spermidine