Control of Rad51 nucleoprotein filament formation in DNA double-strand break repair by homologous recombination

In the yeast Saccharomyces cerevisiae, the genes of the RAD52 epistasis group are required for the repair of DNA double-strand breaks by homologous recombination. Three of these genes, RAD51, RAD55, and RAD57 have been identified as putative RecA homologues. Rad51 is the functional and structural equivalent of RecA and promotes homologous pairing in vitro. This activity is inefficient in the absence of accessory proteins, among which are the other putative RecA homologues, Rad55 and Rad57. A class of rad51 alleles was isolated that suppresses the requirement for RAD55 and RAD57 in DNA repair, but not the other accessory factors. All of the six mutations isolated map to the region of Rad51 that by modeling with RecA corresponds to one of the DNA binding sites. The Rad51-1345T mutant protein shows increased binding to single- and double-stranded DNA, and is proficient in displacement of replication protein A (RPA) from single-stranded DNA, suggesting that the normal function of the Rad55/Rad57 heterodimer is promotion and stabilization of Rad51-ssDNA complexes.; The highest degree of homology between RAD51 and RecA lies in the Walker A and B nucleotide binding motifs. Critical to RecA function is the ability to bind and hydrolyze ATP. Differences seem to exist as to the importance of ATP hydrolysis for Rad51 versus RecA. The mutant Rad51-K191R protein is capable of binding but not hydrolyzing ATP. Study of this mutant protein has suggested that ATP hydrolysis is dispensable for Rad51 function. The purified mutant protein exhibits more stable binding to dsDNA suggesting that the role of ATP hydrolysis for Rad51 is in recycling the protein from double-stranded DNA.; Both the accessory functions of the Rad51 paralogs, Rad55 and Rad57, as well as the ATPase of Rad51 serve to enhance formation of the nucleoprotein filament on ssDNA. ATP hydrolysis releases Rad51 from dsDNA making it available for binding to ssDNA whereas the Rad55/Rad57 paralogs stabilize that initial interaction with ssDNA.