Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.

DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase ␦ (Pol␦) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Pol␦ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Pol and Pol) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Pol␦ during HR.Homologous recombination (HR) is a process that allows genetic exchange between DNA sequences sharing homology and leads to gene conversion or crossovers (COs). The ingenuity of this process is underscored by its conservation from bacteria to humans and its implication in a variety of unrelated nuclear processes. HR is implicated in the restart of stalled replication forks (24). It serves in the repair of DNA damage, such as single-strand gaps, double-strand breaks (DSBs) and interstrand cross-links. It is implicated in mating type switching in yeast strains and in the diversification of immunoglobulinvariable genes in vertebrates (6). In meiosis, the primary function of HR is to establish a physical connection between homologous chromosomes to ensure their correct disjunction at the first meiotic division. In addition, meiotic HR contributes to diversity by creating new linkage arrangements between genes or parts of genes (35).In the past few years, it has become evident that these multiple roles of HR are achieved with variations in the process. Two primary models allow for gene conversion as it is observed in different contexts (Fig. 1). In the seminal DSB repair (DSBR) model of Szostak et al. (41), supported mainly by molecular meiotic studies in yeast, the formation of DSBs is followed by exonucleolytic degradation of the 5Ј ends of the broken duplex to expose single-stranded tails with 3Ј termini (40) (Fig. 1). DSB formation and 5Ј-end resection are followed by the invasion of an intact nonsister chromatid by only one of the two single-stranded tails (13). Single-end invasion (SEI) results in hybrid DNA (hDNA), in which the two strands in a duplex are of different parental origin. If the two parental duplexes are genetically different within the region of strand exchange, the resulting hDNA contains mismatched base pairs...

Homologous recombination between dispersed DNA repeats creates chromosomal rearrangements that are deleterious to the genome. The methylation associated with DNA repeats in many eukaryotes might serve to inhibit homologous recombination and play a role in preserving genome integrity. We have tested the hypothesis that DNA methylation suppresses meiotic recombination in the fungus Ascobolus immersus. The natural process of methylation-induced premeiotically (MIP) was used to methylate the b2 spore color gene, a 7.5-kb chromosomal recombination hot spot. The frequency of crossing-over between two markers flanking b2 was reduced several hundredfold when b2 was methylated on the two homologs. This demonstrates that DNA methylation strongly inhibits homologous recombination. When b2 was methylated on one homolog only, crossing-over was still reduced 50-fold, indicating that the effect of methylation cannot be limited to the blocking of initiation of recombination on the methylated homolog. On the basis of these and other observations, we propose that DNA methylation perturbs pairing between the two intact homologs before recombination initiation and/or impairs the normal processing of recombination intermediates.[Key Words: Ascobolus immersus; chromosome rearrangements; crossing-over; DNA methylation; meiotic recombination; repeat DNA sequences] Received November 28, 1997; revised version accepted February 20, 1998. Crossing-over between dispersed DNA repeats results in chromosomal rearrangements. In eukaryotes, the destructive potential of dispersed repeats through ectopic homologous recombination is well documented (Rouyer et al. 1987;Montgomery et al. 1991;Small et al. 1997). In yeast, artificial duplications placed in ectopic position can interact and generate chromosomal rearrangements through homologous recombination at high frequency during meiosis (Lichten et al. 1987). In higher eukaryotes, the number of repeats per genome is often so high that no single cell would escape genomic rearrangements if ectopic recombination were to occur at high frequencies. Therefore, factors must exist that limit recombination between dispersed repeats. Thuriaux (1977) pointed out that the frequency of crossing-over per unit of physical DNA length decreases with increasing genome size, and proposed that recombination is confined to genes. According to this hypothesis, satellite DNA sequences and interspersed DNA repeats, which constitute the bulk of the intergenic regions, must be poor substrates for meiotic recombination even when in allelic positions. Nucleotidic divergence (Rayssiguier et al. 1989;Radman and Wagner 1993) and an insufficient length of sequence identity (Shen and Huang 1986;Jinks-Robertson et al. 1993) are two factors known to reduce drastically homologous recombination. Nevertheless, other factors that suppress homologous recombination are required to protect genomes against the threat generated by families of long DNA repeats that have diverged little or not at all (e.g., after recent duplication events)....

Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad51, a homolog of the bacterial RecA protein. The nature of these mutations was determined by direct sequencing. The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites. The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis. Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions. By themselves, all the alleles confer a rad51 null phenotype. When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes. This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions.The RAD51 gene of Saccharomyces cerevisiae is involved in recombination and recombinational repair (for a review, see reference 19). It encodes a protein with homologies to the bacterial RecA proteins (1,3,36). These homologies are localized in the regions that form the hydrophobic core of the RecA protein, containing the ATPase and DNA binding domains, as deduced from the three-dimensional structure of the molecule (38-40).Rad51 forms with double-stranded (ds) DNA (27) and single-stranded (ss) DNA (42) filaments similar in structure to those observed with RecA. In the presence of the Rpa DNAbinding proteins, it catalyzes the homologous DNA strand exchange reaction (41,42). Three other S. cerevisiae genes, RAD55 (20), RAD57 (16), and DMC1 (7), also code for proteins with homologies to the ATPase domain of RecA. RAD51, RAD55, and RAD57 belong to the RAD52 epistasis group of genes involved in the repair of ionizing radiation (10) and are part of the same homologous recombination pathway (29). These proteins may well be part of a multiprotein complex: Rad51 interacts with Rad52 in vitro (36) and in vivo (24); it also interacts with Rad55, which in turn interacts with Rad57 (11,15). DMC1 is expressed only during meiosis and is involved in meiotic recombination (7). The Dmc1 and Rad51 proteins appear to be associated in recombination complexes (6). Homologs of Rad51 have also been identified in two other yeasts, Kluyveromyces lactis (9) and Schizosaccharomyces pombe (14, 35), in Neurospora crassa (8), and in higher eucaryotes, including Xenopus laevis (21), chicken (5), mouse (26,35), and human cells (35). In all cases, the regions of greatest homology are confined to the ATPase and DNA binding domains. The human protein forms nucleofilaments with both ss and ds DNAs (4).In ...

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase ␦, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.H OMOLOGOUS recombination plays a critical role parental origin. If the two parental duplexes are genetically different within the region of strand exchange, the in maintaining genome integrity throughout cell division. In meiosis, homologous recombination is esresulting hDNA contains mismatched base pairs and is referred to as heteroduplex DNA. sential for proper homolog pairing and for the correct segregation of chromosomes at the first meiotic division.Single-end invasion intermediates can be channeled toward either of two repair pathways. In the first pathIn vegetative cells, recombination plays an important role during DNA replication by providing a mechanism way, described by the DSB repair model (Szostak et al. 1983;Sun et al. 1989), DNA synthesis, capture of the to bypass DNA lesions and other obstacles that block replication fork progression. Homologous recombinasecond end, and ligation generate a double Holliday junction intermediate with asymmetric hDNA (i.e., hDNA on tion also provides a means to generate new combinations of genetic markers through gene conversion and only one of the two duplexes) on each side of the DSB and on each chromatid (Figure 1, left). If a Holliday crossing over, thereby generating genetic diversity among different individuals in the same population. junction undergoes branch migration, then hDNA will be formed on both duplexes (symmetric hDNA). EvenNumerous insights into the mechanism of homologous recombination have been obtained from meiotic studies tually, double Holliday junction intermediates are resolved by cutting, at each junction, either both outside using the convenient model organism, Saccharomyces cerevisstrands or both inside strands. Cutting of the two junciae (Paques and Haber 1999). Meiotic recombination in tions in opposite directions generates crossovers, while budding yeast is initiated by the formation of DNA doublecutting in the same direction generates noncrossovers. strand breaks (DSBs) at recombination hotspots. The The second pathway is described by the synthesisstrands with 5Ј ends at the site of the break are processed dependent strand annealing (SDSA) model (Paques to expose single-stranded tails with 3Ј termini (Figure and Haber 1999) and supported by recent meiotic stud-1). DSB formation and 5Ј-end resection are followed by ies (

The transfer of methylation between alleles represents a plausible epigenetic mutational mechanism to explain loss of imprinting in mammals and paramutation in plants. Here, we have exploited advantages unique to the fungus Ascobolus immersus to obtain direct experimental evidence that methylation transfer can occur between homologous chromosomes. A methylated allele and an unmethylated allele of the Ascobolus b2 spore color gene were brought together in individual meiotic cells. Frequent transfer of methylation to the unmethylated allele was observed. This transfer was polarized 5' to 3' along the b2 gene, as is gene conversion, and always accompanied the latter process when tested in the same cross. These and other observations strongly suggest that methylation transfer and recombination are mechanistically related.

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.

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.