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.     

Stress Activated Protein Kinase Pathway Modulates Homologous Recombination in Fission Yeast

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.     

Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae

Agrobacterium tumefaciens delivers transferred DNA (T-DNA) into cells of plants and yeast. In plants, the T-DNA integrates at random positions into the genome by non-homologous recombination (NHR), whereas in yeast the T-DNA preferably integrates by homologous recombination (HR). Here we show that T-DNA integration by HR in yeast requires the recombination/repair proteins Rad51 and Rad52, but not Rad50, Mre11, Xrs2, Yku70 and Lig4. In the HR events a remarkable shift from insertion-type events to replacement events was observed in rad50, mre11 and xrs2 mutants. Residual integration in the rad51 mutant occurred predominantly by HR, whereas in the rad52 mutant integration occurred exclusively by NHR. Previously, we found that T-DNA integration by NHR is abolished in a yku70 mutant. Thus, Rad52 and Yku70 are the key regulators of T-DNA integration, channeling integration into either the HR or NHR pathway.     

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.     

Rad51 Paralog Complexes BCDX2 and CX3 Act at Different Stages in the BRCA1-BRCA2-Dependent Homologous Recombination Pathway

The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.     

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a ‘protein delivery approach’ offers a new tool for developing novel strategies for genome modification and gene therapy applications.     

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.     

Homologous recombination is required for AAV-mediated gene targeting

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ~5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.     

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.     

Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity

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.     

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.     

Characterization of Rad51 from Apicomplexan Parasite Toxoplasma gondii: An Implication for Inefficient Gene Targeting

Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite Toxoplasma gondii. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher K(M) and much lower k(cat) compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in T. gondii but also could be exploited to facilitate high throughput knockout as well as epitope tagging of Toxoplasma genes.     

Role of the Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

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 amino-terminal 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.     

Tumor suppressor RecQL5 controls recombination induced by DNA crosslinking agents

RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA (ssDNA) filaments in vitro. However, the precise roles of RecQL5 in the cell remain elusive. Here, we present evidence suggesting that RecQL5 is involved in DNA interstrand crosslink (ICL) repair. Chicken DT40 RECQL5 gene knockout (KO) cells showed sensitivity to ICL-inducing agents such as cisplatin (CDDP) and mitomycin C (MMC) and a higher number of chromosome aberrations in the presence of MMC than wild-type cells. The phenotypes of RECQL5 KO cells resembled those of Fanconi anemia gene KO cells. Genetic analysis using corresponding gene knockout cells showed that RecQL5 is involved in the FANCD1 (BRCA2)-dependent ICL repair pathway in which Rad51-ssDNA filament formation is promoted by BRCA2. The disappearance but not appearance of Rad51-foci was delayed in RECQL5 KO cells after MMC treatment. Deletion of Rad54, which processes the Rad51-ssDNA filament in HR, in RECQL5 KO cells increased sensitivity to CDDP and further delayed the disappearance of Rad51-foci, suggesting that RecQL5 and Rad54 have different effects on the Rad51-ssDNA filament. Furthermore, the frequency and variation of CDDP-induced gene conversion at the immunoglobulin locus were increased in RECQL5 KO cells. These results suggest that RecQL5 plays a role in regulating the incidence and quality of ICL-induced recombination.     

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.     

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.     

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.     

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.     

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.