Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.

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Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation.

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Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.Saccharomyces cerevisiae genes of the RAD52 epistasis group, viz, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11, and XRS2, are required for genetic recombination and DNA double-strand break repair by recombination. Since genetic recombination is indispensable for the disjunction of homologous chromosomal pairs during meiosis I, mutational inactivation of the RAD52 group genes engenders severe meiotic defects, manifest as a failure to sporulate and spore inviability (1-5). The results from recent cloning studies have revealed a remarkable degree of conservation of the RAD52 group genes among eukaryotes, from yeast to humans.A conceptual model concerning the mechanism of homologous recombination has been formulated based on genetic studies in S. cerevisiae (6). When S. cerevisiae cells enter meiosis, DNA double-strand breaks are formed at various chromosomal "hot spots" that exhibit a propensity to recombine. Subsequent to break formation, unidirectional nucleolytic end-processing of the break yields 3Ј ssDNA 1 tails of a considerable length (7,8).It is believed that nucleation of various recombination factors onto the ssDNA tails gives rise to a nucleoprotein complex that has the ability to conduct a search to locate a DNA homolog and to invade the homolog to form heteroduplex DNA. Concurrent and subsequent events include DNA synthesis to replace the genetic information eliminated during double-strand break processing, resolution of the DNA intermediates, and DNA ligation to complete the recombination process. The repair of DNA double-strand breaks induced by ionizing radiation and radiomimetic chemicals very likely proceeds through the same mechanistic route, as the repair process shares the same requirement for the RAD52 epistasis group genes. Extensive genetic evidence has indicated that the nucleolytic end-processing of DNA double-strand breaks during recombination processes is dependent on the RAD50, MRE11, and XRS2 genes. The Mre11 protein from both yeast (9 -11) and humans (12) and a protein complex (13) consisting of the human Rad50, Mre11, and the Xrs2 equival...