Summary Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here we show Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homologue Uls1, accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, non–damage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci and Rdh54 predominates for removal of non-damage-associated foci.

The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. Here we describe an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, we demonstrate that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. We furnish evidence that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54⌬ mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54⅐Rad51 complex formation. The Rdh54 expression and purification procedures described here should facilitate the functional dissection of this DNA recombination/repair factor. Homologous recombination (HR)3 helps eliminate deleterious lesions, including DNA strand breaks and interstrand cross-links, from chromosomes. Furthermore, by linking homologous chromosomes through crossover formation, HR helps ensure the proper segregation of the chromosomes in the first meiotic division. Interestingly, HR can also provide a means of elongating shortened telomeres in the absence of telomerase. Because of its involvement in various aspects of chromosome maintenance, mutants of HR typically accumulate chromosome aberrations and exhibit a mutator phenotype. In fact, in higher eukaryotes, deletion of HR genes often engenders cell inviability, believed to reflect the requirement of HR for cells to successfully complete DNA replication during the S phase (1). That HR is critical for genome stability is underscored by the realization that the cancer-prone diseases Fanconi anemia and Bloom's syndrome exhibit either HR deficiency or HR deregulation, respectively (2, 3). Furthermore, cells deficient in the tumor suppressors BRCA1 and BRCA2 are marked by a pronounced HR defect (4 -7). The linkage of HR impairment or deregulation to the cancer phenotype emphasizes the importance of delineating the mechanistic underpinnings of the HR machinery.The genetic requirement of HR was initially defined in the budding y...

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Ribonucleotides can become embedded in DNA from insertion by DNA polymerases, failure to remove Okazaki fragment primers, R-loops that can prime replication, and RNA/cDNA-mediated recombination. RNA:DNA hybrids are removed by RNase H enzymes. Single rNMPs in DNA are removed by RNase H2 and if they remain on the leading strand, can lead to mutagenesis in a Top1-dependent pathway. rNMPs in DNA can also stimulate genome instability, among which are homologous recombination gene conversion events. We previously found that, similar to the rNMP-stimulated mutagenesis, rNMP-stimulated recombination was also Top1-dependent. However, in contrast to mutagenesis, we report here that recombination is not stimulated by rNMPs incorporated by the replicative polymerase epsilon. Instead, recombination seems to be stimulated by multiple contiguous rNMPs, which may arise from R-loops or replication priming events.

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair.