Yeast Rad54 Promotes Rad51-dependent Homologous DNA Pairing via ATP Hydrolysis-driven Change in DNA Double Helix Conformation

Abstract

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...

A Novel Function of Rad54 Protein

“…Stabilization of the Rad51-DNA Complex Is Independent of the ATPase Activity of Rad54 Protein-Previously, it was shown that the Rad54 K341R mutant protein, which lacks ATP hydrolysis and dsDNA unwinding activities, is still proficient in binding to both DNA and Rad51 protein (29). 2 Therefore, we tested this mutant protein with regard to its capacity to stabilize Rad51 nucleoprotein filaments.…”

“…It was found that, in contrast to other proteins that stimulate the DNA pairing activity of Rad51 protein, Rad54 protein acts at a later (synaptic) stage of DNA strand exchange (24,29). Rad54 protein belongs to the Snf2/Swi2 group of proteins, which are involved in ATP-dependent remodeling of protein complexes, including mainly chromatin complexes (30).…”

Hed1 regulates Rad51-mediated recombination via a novel mechanism

“…The Rad54 DNA supercoiling activity can be conveniently monitored by a topoisomerase-linked assay (Petukhova et al 1999;Van Komen et al 2000). Specifically, the negative supercoils generated by Rad54 are removed by Escherichia coli topoisomerase I to yield overwound (OW) DNA (Fig.…”

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

“…The promotion of Rad51-mediated homologous DNA pairing and strand exchange by Rad54 requires the ATPase activity of the latter and physical interaction between these two HR factors [27][28][29]. Interestingly, Rad54 dissociates Rad51 from duplex DNA via its DNA translocase activity, a function believed to be important for the intracellular recycling of Rad51 and possibly for freeing the primer end in the D-loop structure to initiate the DNA synthesis reaction [30].…”