Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.     

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.     

Rad51-independent interchromosomal double-strand break repair by gene conversion requires Rad52 but not Rad55, Rad57, or Dmc1

Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.     

Functional analysis of the Drosophila Rad51 gene (spn-A) in repair of DNA damage and meiotic chromosome segregation

Rad51 is a crucial enzyme in DNA repair, mediating the strand invasion and strand exchange steps of homologous recombination (HR). Mutations in the Drosophila Rad51 gene (spn-A) disrupt somatic as well as meiotic double-strand break (DSB) repair, similar to fungal Rad51 genes. However, the sterility of spn-A mutant females prevented a thorough analysis of the role of Rad51 in meiosis. In this study, we generated transgenic animals that express spn-A dsRNA under control of an inducible promoter, and examined the effects of inhibiting expression of spn-A on DNA repair, meiotic recombination and meiotic chromosome pairing and segregation. We found that depletion of spn-A mRNA had no effect on the viability of non-mutagen-treated transgenic animals but greatly reduced the survival of larvae that were exposed to the radiomimetic drug MMS, in agreement with the MMS and X-ray sensitivity of spn-A mutant animals. We also found that increases in dose of spn-A gene enhanced larval resistance to MMS exposure, suggesting that at high damage levels, Rad51 protein levels may be limiting for DNA repair. spn-A RNAi strongly stimulated X-X nondisjunction and decreased recombination along the X in female meiosis, consistent with a requirement of Rad51 in meiotic recombination. However, neither RNAi directed against the spn-A mRNA nor homozygosity for a spn-A null mutation had any effect on male fertility or on X-Y segregation in male meiosis, indicating that Rad51 likely plays no role in male meiotic chromosome pairing. Our results support a central role for Rad51 in HR in both somatic and meiotic DSB repair, but indicate that Rad51 in Drosophila is dispensable for meiotic chromosome pairing. Our results also provide the first demonstration that RNAi can be used to inhibit the functions of meiotic genes in Drosophila.     

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.     

RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens

Gene targeting (GT) is a major tool for basic and applied research during which the transforming DNA, which shares sequence homology with a chromosomal target, integrates at the corresponding locus by homologous recombination (HR). In eukaryotes, GT recruits enzymes from the HR-mediated double strand break repair pathway. Different mechanisms of HR have been described which depend on the Rad52 epistasis group of genes, but which specific mechanism is used by the cell for GT remains unclear. In Saccharomyces cerevisiae, the RAD52 protein is essential for GT, and the RAD51 protein plays a minor role. In filamentous fungi and animal cells, however, GT depends on RAD51 and is weakly affected by suppression of RAD52. Genetic evidence also indicates that the non-homologous end-joining pathway of DSB repair has a negative impact on GT efficiencies, but how the balance between these two pathways is controlled is poorly understood. Here, we have examined the role of RAD51 in the only plant that exhibits high GT frequencies, the model bryophyte Physcomitrella patens. Our results show that the two RAD51 proteins have partially redundant functions in the maintenance of genome integrity and resistance to ionizing radiation. Furthermore, we demonstrate that loss of function of the two RAD51 proteins completely abolishes GT and strongly increases illegitimate integration rates in this moss. These findings demonstrate for the first time in plant the critical role of RAD51 in controlling the balance between targeted and random integration events observed upon transgenesis, and confirm that P. patens is a particularly interesting tool for studying GT in higher eukaryotes.     

Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line

Overexpression of the RAD52 epistasis group of gene products is a convenient way to investigate their in vivo roles in homologous recombination (HR) and DNA repair. Overexpression has the further attraction that any associated stimulation of HR may facilitate gene-targeting applications. Rad51p or Rad52p overexpression in mammalian cells have previously been shown to enhance some forms of HR and resistance to ionising radiation, but the effects of Rad52p overexpression on gene targeting have not been tested. Here we show that Rad52p overexpression inhibits gene targeting while stimulating extrachromosomal HR. We also find that Rad52p overexpression affects cell-cycle distribution, impairs cell survival and is lost during extensive passaging. Therefore, we suggest that excess Rad52p can inhibit the essential RAD51-dependent pathways of HR most likely to be responsible for gene targeting, while at the same time stimulating the RAD51-independent pathway thought to be responsible for extrachromosomal HR. The data also argue against Rad52p overexpression as a means of promoting gene targeting, and highlight the limitations of using a single HR assay to assess the overall status of HR.     

Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52^−/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52^−/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52^−/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52^−/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Role of Elg1 protein in double strand break repair

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G₂/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR.     

Rad51 Inhibits Translocation Formation by Non-Conservative Homologous Recombination in Saccharomyces cerevisiae

Chromosomal translocations are a primary biological response to ionizing radiation (IR) exposure, and are likely to result from the inappropriate repair of the DNA double-strand breaks (DSBs) that are created. An abundance of repetitive sequences in eukaryotic genomes provides ample opportunity for such breaks to be repaired by homologous recombination (HR) between non-allelic repeats. Interestingly, in the budding yeast, Saccharomyces cerevisiae the central strand exchange protein, Rad51 that is required for DSB repair by gene conversion between unlinked repeats that conserves genomic structure also suppresses translocation formation by several HR mechanisms. In particular, Rad51 suppresses translocation formation by single-strand annealing (SSA), perhaps the most efficient mechanism for translocation formation by HR in both yeast and mammalian cells. Further, the enhanced translocation formation that emerges in the absence of Rad51 displays a distinct pattern of genetic control, suggesting that this occurs by a separate mechanism. Since hypomorphic mutations in RAD51 in mammalian cells also reduce DSB repair by conservative gene conversion and stimulate non-conservative repair by SSA, this mechanism may also operate in humans and, perhaps contribute to the genome instability that propels the development of cancer.     

Fission Yeast Rad52 Phosphorylation Restrains Error Prone Recombination Pathways

Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.     

Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo

Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene ~3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast.     

Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.     

INO80-dependent chromatin remodeling regulates early and late stages of mitotic homologous recombination

Chromatin remodeling is emerging as a critical regulator of DNA repair factor access to DNA damage, and optimum accessibility of these factors is a major determinant of DNA repair outcome. Hence, chromatin remodeling is likely to play a key role in genome stabilization and tumor suppression. We previously showed that nucleosome eviction near double-strand breaks (DSBs) in yeast is regulated by the INO80 nucleosome remodeling complex and is defective in mutants lacking the Arp8 subunit of INO80. In the absence of homologous donor sequences, RPA recruitment to a DSB appeared normal in arp8Δ, but Rad51 recruitment was defective. We now show that the early strand invasion step of homologous recombination (HR) is markedly delayed in an arp8Δ haploid, but there is only a minor defect in haploid HR efficiency (MAT switching). In an arp8Δ diploid, interhomolog DSB repair by HR shows a modest defect that is partially suppressed by overexpression of Rad51 or its mediator, Rad52. In wild type cells, DSB repair typically results in gene conversion, and most gene conversion tracts are continuous, reflecting efficient mismatch repair of heteroduplex DNA. In contrast, arp8Δ gene conversion tracts are longer and frequently discontinuous, indicating defects in late stages of HR. Interestingly, when a homologous donor sequence is present, Rad51 is recruited normally to a DSB in arp8Δ, but its transfer to the donor is delayed, and this correlates with defective displacement of donor nucleosomes. We propose that retained nucleosomes at donors destabilize heteroduplex DNA or impair mismatch recognition, reflected in delayed strand invasion and altered conversion tracts.     

Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.     

DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks (DSBs) in mitotic and meiotic cells. HR occurs through a series of steps involving DSB resection, invasion of single-stranded DNA into homologous duplex DNA to form a D loop, repair synthesis, and second-end capture. We show that DNA repair synthesis, catalyzed by human DNA polymerase eta (poleta) acting upon the priming strand of a D loop, leads to capture and annealing of the second end of a resected DSB in reactions mediated by RAD52 protein. Second-end capture products were not detected when poleta was replaced by other polymerases such as poldelta or poliota. RAD52 could not be replaced by RAD51. We also found that the RAD52-dependent reaction was stimulated by the single-strand binding protein RPA, but not by E. coli SSB. Following repair synthesis and second-end capture, de novo DNA synthesis was observed from the captured second DNA end.     

Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths

DNA double-strand breaks (DSBs) in yeast are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad51 forms nucleoprotein filaments at processed broken ends that effect strand exchange, forming heteroduplex DNA (hDNA) that gives rise to a gene conversion tract. We hypothesized that excess Rad51 would increase gene conversion tract lengths. We found that excess Rad51 reduced DSB-induced HR but did not alter tract lengths or other outcomes including rates of crossovers, break-induced replication, or chromosome loss. Thus, excess Rad51 appears to influence DSB-induced HR at an early stage. MAT heterozygosity largely mitigated the inhibitory effect of excess Rad51 on allelic HR, but not direct repeat HR. Excess Rad52 had no effect on DSB-induced HR efficiency or outcome, nor did it mitigate the dominant negative effects of excess Rad51. Excess Rad51 had little effect on DSB-induced lethality in wild-type cells, but it did enhance lethality in yku70Delta mutants. Interestingly, dnl4Delta showed marked DSB-induced lethality but this was not further enhanced by excess Rad51. The differential effects of yku70Delta and dnl4Delta indicate that the enhanced killing with excess Rad51 in yku70Delta is not due to its NHEJ defect, but may reflect its defect in end-protection and/or its inability to escape from checkpoint arrest. Srs2 displaces Rad51 from nucleoprotein filaments in vitro, suggesting that excess Rad51 might antagonize Srs2. We show that excess Rad51 does not reduce survival of wild-type cells treated with methylmethane sulfonate (MMS), or cells suffering a single DSB. In contrast, excess Rad51 sensitized srs2Delta cells to both MMS and a single DSB. These results support the idea that excess Rad51 antagonizes Srs2, and underscores the importance of displacing Rad51 from nucleoprotein filaments to achieve optimum repair efficiency.     

Midgestation lethality in mice deficient for the RecA-related gene, Rad51d/Rad51l3

Homologous recombination (HR) occurs in all organisms, and is important for repair of DNA damage, chromosome segregation during meiosis, and genetic diversification. Genes critical for recombinational DNA repair and meiotic recombination include members of the RecA/RAD51 family, of which seven have been identified in mammals. Here, we describe the disruption of Rad51d (recently designated Rad51l3) in mice and its phenotypic consequences. Rad51d-deficient mice die between 8.5 and 11.5 dpc. The affected embryos are smaller than littermates, posteriorly truncated, and developmentally delayed. Embryonic fibroblasts from mutant embryos could not be propagated more than one generation in culture. Rad51d-deficient blastocysts were not sensitive to gamma radiation or methylmethanesulfonate (MMS) in blastocyst outgrowth experiments. The variable and generalized developmental progression defects in Rad51d-deficient embryos suggests that mutant cells may undergo delayed or suboptimal repair of DNA damage, resulting in accumulated degrees of mutation and/or cell cycle perturbation that are incompatible with normal embryonic development. genesis 26:167-173, 2000.