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Papers In Press, published online ahead of print February 11, 2002
Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, PA 19107
Corresponding Author: rfishel{at}lac.jci.tju.edu
RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. 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/ATPgS. hRAD51 fully saturates with ATP/ATPgS regardless of DNA cofactor (KD = 5mM; 1 ATP : 1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPgS binding (Kapp1 = 3mM; 1 ADP : 1 hRAD51), while a second mode occurred at elevated ADP concentrations (Kapp2 * 125mM; >1 ADP : 1 hRAD51). We could detect ADPýATP exchange in the high-affinity ADP binding mode (Kapp1), but not the low-affinity binding mode (Kapp2). At low ATP concentrations (<0.3 mM), ADP and ATPgS competitively inhibit the hRAD51 ATPase (Kmapp> Km). However, at high ATP (>0.3 mM), the hRAD51 ATPase was stimulated by concentrations of ATPgS that were 20-fold above the KD. Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially (~10-fold) and ATP modestly (~3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament likely restricts the hRAD51 ATPase.
J. Biol. Chem, 10.1074/jbc.M109916200
Submitted on October 12, 2001
Revised on February 7, 2002
Accepted on February 10, 2002
Biochemical characterization of the human RAD51 Protein: II. Adenosine nucleotide binding and competition
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