Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination.[Keywords: Regulation of meiotic recombination; interhomolog crossovers; Rad51 recombinase; double-strand break repair; homologous recombination] Supplemental material is available at http://www.genesdev.org.

Homologous recombination (HR) is a major DNA repair pathway and therefore essential for maintaining the integrity of the genome. HR is catalyzed by proteins encoded by genes of the RAD52 epistasis group, including the recombinase Rad51 and its mediator Rad52. HR proteins fused with green fluorescent protein form foci at damaged DNA reflecting the assembly of repair centers that harbor a high concentration of repair proteins. Rad52 mediates the recruitment of Rad51 and other HR proteins to DNA damage. To understand the mechanism for the assembly of Rad52-dependent DNA repair centers, we used a mutational strategy to identify a Rad52 domain essential for its recruitment to DNA repair foci. We present evidence to implicate an acidic domain in Rad52 in DNA repair focus formation. Mutations in this domain confer marked DNA damage sensitivity and recombination deficiency. Importantly, these Rad52 mutants are specifically compromised for interaction with the single-stranded DNA-binding factor RPA. Based on these findings, we propose a model where Rad52 displaces RPA from single-stranded DNA using the acidic domain as a molecular lever.In eukaryotes, DNA double strand break (DSB) 4 repair is essential for maintaining genetic stability. The major pathway of DSB repair in the yeast Saccharomyces cerevisiae is homologous recombination (HR), and the evolutionarily conserved proteins involved in this process are encoded by members of the RAD52 epistasis group, including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, RFA1, MRE11, and XRS2 (1). In mitotic cells, most DSBs are eliminated by the synthesis-dependent strand annealing pathway of HR (2). The first step of this pathway involves resection of the ends at the DSB to produce a pair of 3Ј-single-stranded tails. Subsequently, one of these single-stranded tails invades an intact homologous double-stranded DNA sequence to produce a D-loop. DNA polymerase extends the invading end, hence acquiring DNA information that is complementary to the noninvading end. At this stage, the invading strand is dissociated from the invaded duplex and anneals to the noninvading end of the break. Repair is completed when the break is sealed via additional DNA synthesis and ligation.In vitro it has been shown that Rad51 can catalyze the DNA strand invasion reaction via a filamentous intermediate called the presynaptic filament (3, 4). The efficiency of this reaction is dependent on several accessory factors, including the heterotrimeric single-stranded DNA-binding protein RPA (3, 5-7). RPA minimizes intramolecular secondary structure in the singlestranded DNA (ssDNA) that would otherwise impede presynaptic filament assembly. Paradoxically, if an amount of RPA sufficient to saturate the ssDNA is added to the in vitro recombination reaction prior to or together with Rad51, it strongly inhibits strand invasion by limiting access of Rad51 to the ssDNA (1, 8). Chromatin immunoprecipitation and cytological studies have also shown that RPA excludes Rad51 from the HR substrate (9, 10 -13...

A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function. Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNAbound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad52 in the rad52 ⌬327 background enhances the association of Rad51 protein with a HO-made DNA double-strand break and partially complements the methylmethane sulfonate sensitivity of the mutant cells. Our results provide a mechanistic framework for rationalizing the multi-faceted role of Rad52 in recombination and DNA repair.

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised aminoterminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous recombination. Homologous recombination (HR)2 represents an error-free pathway for the elimination of DNA breaks and DNA interstrand cross-links from chromosomes, and it also provides a means for the restart of stalled or damaged DNA replication forks. Moreover, HR plays a crucial role in the proper segregation of homologous chromosomes in meiosis I (for review, see Refs. 1 and 2). Accordingly, a deficiency in HR or its deregulation leads to meiotic aneuploidy, genomic instability, and the tumor phenotype (for review, see Refs. 1-3).HR is often initiated via the formation of DNA double-strand breaks (DSBs). After DSB formation, the ends of the break are nucleolytically processed to yield single-stranded DNA (ssDNA) tails (for review, see Ref. 4). The ubiquitous singlestrand DNA-binding protein RPA first decorates the ssDNA. RPA is subsequently replaced by the Rad51 recombinase to form a right-handed helical polymer, referred to as the presynaptic filament. This nucleoprotein complex conducts a search for a homologous DNA sequence and invades the latter to form a nascent DNA joint called the displacement loop, or D-loop (for review, see Ref. 5). After invasion, the 3Ј end of the invading strand primes DNA synthesis, which serves to replace the genetic information lost during the end-processing reaction and to expand the D-loop.The D-loop intermediate can be resolved by two mechanistically distinct pathways. In the synthesis-dependent singlestrand annealing pathway, the invading DNA strand with its newly synthesized DNA extension is displaced from the information donor and subsequently annealed to the ssDNA tail that adjoins the second end of the break. The synthesis-dependent single-strand annealing mode of HR does not have the potential of generating crossover recombinants. In the DNA doublestrand break repair pathway, the D-loop is maintained and recruits the second ssDNA tail by annealing, to assemble a double Holliday junction. The resolution of the double Holliday junction by a resolvase then yields a mixture of crossover and noncrossover recombinants (1).The Rad52 protein of Saccharomyces cerevisiae is needed for two di...