BINDING OF RAD51P TO DNA - INTERACTION OF RAD5LP WITH SINGLE-STRANDEDAND DOUBLE-STRANDED DNA

Like RecA, Saccharomyces cerevisiae Rad51p promotes strand exchange be tween circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). We have investigated several parameters characteristic of the interaction of Rad51p with ssDNA and dsDNA, particularly the effe cts of the nucleotide cofactors ATP and ADP and the analogs adenosine 5'-O-(thio-triphosphate) (ATP gamma S) and adenylyl-imidodiphosphate ( AMP-PNP). Rad51p binding to both 1-N-6-ethenoadenosine and 3-N-4-ethen ocytidine ssDNA (epsilon DNA) and dsDNA requires the presence of Mg2and ATP; no binding occurs in the presence of ADP, AMP-PNP, or ATP gam ma S. Binding of Rad51p to dsDNA also requires ATP; ADP is ineffective , whereas ATP gamma S and AMP-PNP are considerably less able to promot e binding and only at elevated concentrations of Rad51p, ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. The K-d. va lues for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 mu M, respectively. Rad51p binding occurs with a stoichiometry o f one monomer of Rad51p per similar to 6.3 nucleotides of epsilon DNA and similar to 3.3 base pairs of dsDNA. Once formed. Rad51p ssDNA comp lexes are stable so long as sufficient ATP levels are maintained. ATP hydrolysis causes dissociation of Rad51p from DNA. Moreover, the prefo rmed complex is stable in the presence of a 10-fold excess of ADP or A MP-PNP over ATP. ATP gamma S, however, in the same-fold excess over AT P causes dissociation of the Rad51p ssDNA complex.