High-Resolution Mapping of Two Types of Spontaneous Mitotic Gene Conversion Events in Saccharomyces cerevisiae

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution.     

P-Element-Induced Male Recombination and Gene Conversion in Drosophila

A P-element insertion flanked by 13 restriction fragment length polymorphism (RFLP) marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the nonrearranged male recombinants. Most recombination events can be explained by the ``hybrid element insertion' model, but, for those lacking a duplication or deletion, a second step involving double-strand gap repair must be postulated to explain the distribution of crossover points.     

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.     

Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but approximately 22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n - 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.     

Use of a Chromosomal Inverted Repeat to Demonstrate That the Rad51 and Rad52 Genes of Saccharomyces Cerevisiae Have Different Roles in Mitotic Recombination

An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 X 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 had functions in addition to those of the Rad51/Rad52 protein complex.     

Interchromosomal crossover in human cells is associated with long gene conversion tracts

Crossovers have rarely been observed in specific association with interchromosomal gene conversion in mammalian cells. In this investigation two isogenic human B-lymphoblastoid cell lines, TI-112 and TSCER2, were used to select for I-SceI-induced gene conversions that restored function at the selectable thymidine kinase locus. Additionally, a haplotype linkage analysis methodology enabled the rigorous detection of all crossover-associated convertants, whether or not they exhibited loss of heterozygosity. This methodology also permitted characterization of conversion tract length and structure. In TI-112, gene conversion tracts were required to be complex in tract structure and at least 7.0 kb in order to be selectable. The results demonstrated that 85% (39/46) of TI-112 convertants extended more than 11.2 kb and 48% also exhibited a crossover, suggesting a mechanistic link between long tracts and crossover. In contrast, continuous tracts as short as 98 bp are selectable in TSCER2, although selectable gene conversion tracts could include a wide range of lengths. Indeed, only 16% (14/95) of TSCER2 convertants were crossover associated, further suggesting a link between long tracts and crossover. Overall, these results demonstrate that gene conversion tracts can be long in human cells and that crossovers are observable when long tracts are recoverable.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.     

Length and Distribution of Meiotic Gene Conversion Tracts and Crossovers in Saccharomyces Cerevisiae

We have measured gene conversion tract length in strains of the yeast Saccharomyces cerevisiae containing three to six restriction site heterozygosities in a 9-kb interval. Tetrads containing a conversion were identified genetically by nonmendelian segregation of a marker in the middle of the interval. Gene conversions accompanied by a crossover have a tract length of 1.4 kb +/- 0.7 kb, which is indistinguishable from a tract length of 1.6 +/- 0.8 for conversions without an associated exchange. Among tetrads identified first as having a crossover in the interval, the average gene conversion tracts were apparently significantly shorter (0.71 +/- 1). We provide evidence that this apparent difference is due to the method of measuring conversion tracts and does not reflect a real difference in tract length. We also provide evidence that the number and position of restriction site markers alters the apparent distribution of the conversion tracts. More than ninety percent of the conversion tracts spanning three or more sites were continuous.     

Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity

RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.     

The Role of Exo1p Exonuclease in DNA End Resection to Generate Gene Conversion Tracts in Saccharomyces cerevisiae

The yeast Exo1p nuclease functions in multiple cellular roles: resection of DNA ends generated during recombination, telomere stability, DNA mismatch repair, and expansion of gaps formed during the repair of UV-induced DNA damage. In this study, we performed high-resolution mapping of spontaneous and UV-induced recombination events between homologs in exo1 strains, comparing the results with spontaneous and UV-induced recombination events in wild-type strains. One important comparison was the lengths of gene conversion tracts. Gene conversion events are usually interpreted as reflecting heteroduplex formation between interacting DNA molecules, followed by repair of mismatches within the heteroduplex. In most models of recombination, the length of the gene conversion tract is a function of the length of single-stranded DNA generated by end resection. Since the Exo1p has an important role in end resection, a reduction in the lengths of gene conversion tracts in exo1 strains was expected. In accordance with this expectation, gene conversion tract lengths associated with spontaneous crossovers in exo1 strains were reduced about twofold relative to wild type. For UV-induced events, conversion tract lengths associated with crossovers were also shorter for the exo1 strain than for the wild-type strain (3.2 and 7.6 kb, respectively). Unexpectedly, however, the lengths of conversion tracts that were unassociated with crossovers were longer in the exo1 strain than in the wild-type strain (6.2 and 4.8 kb, respectively). Alternative models of recombination in which the lengths of conversion tracts are determined by break-induced replication or oversynthesis during strand invasion are proposed to account for these observations.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

M26 Recombinational Hotspot and Physical Conversion Tract Analysis in the Ade6 Gene of Schizosaccharomyces Pombe

At the ade6 locus of Schizosaccharomyces pombe flanking markers have been introduced as well as five silent restriction site polymorphisms: four in the 5' upstream region and one in the middle of the gene. The mutations ade6-706, ade6-M26 (both at the 5' end) and ade6-51 (middle of the gene) were used as partners for crosses with the 3' mutation ade6-469. From these three types of crosses, wild-type recombinants were selected and analyzed genetically to assess association with crossing-over and physically to determine conversion tract lengths. The introduced restriction site polymorphisms (five vs. only one) neither influenced the pattern of recombinant types nor the distribution of conversion tracts. The hotspot mutation M26 enhances crossing-over and conversion to the same proportion. M26 not only stimulates conversion at the 5' end, but does this also (to a lower extent) at the 3' end of ade6 at a distance of more than 1 kb. The majority of meiotic conversion tracts are continuous and postmeiotic segregation of polymorphic sites is rare. Conversion tracts are slightly shorter with M26 in comparison with its control 706. The mean minimal length of tracts varies from 670 bp (M26) to 890 bp (706) to 1290 bp (51). It is concluded that M26 acts as an initiation site of recombination or enhances initiation of recombination. M26 does not act by termination of conversion. A region of recombination initiation exists at the 5' end of the ade6 gene also in the absence of the ade6-M26 hotspot mutation.     

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.     

Analysis of spontaneous gene conversion tracts within and between mammalian chromosomes

In the present study, we report the first characterization of gene conversion tract length, continuity and fidelity for pathways of gene targeting, ectopic and intrachromosomal homologous recombination using the same locus and mammalian somatic cell type. In this isogenic cell system, the vast majority of recombinants (> 97%) are generated by homologous recombination and display a high degree of fidelity in the gene conversion process. Individual gene conversion tracts are highly likely to involve single, independent recombination events and proceed through a heteroduplex DNA intermediate. In all recombination pathways, gene conversion tracts are long, extending up to ∼ 2 kb. Most gene conversion tracts are continuous in favor of donor region sequences, but in a small fraction of recombinants (15%), discontinuous gene conversion tracts are observed. In most cases, the recombination donor sequence is unaltered, although in two cases of intrachromosomal recombination, both recombination donor and recipient sequences bear gene conversion tracts. Overall, gene conversion events are similar, both qualitatively and quantitatively, for homologous recombination within and between mammalian chromosomes.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast.

Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.     

Characterization of the Roles of the Saccharomyces Cerevisiae Rad54 Gene and a Homologue of Rad54, Rdh54/Tid1, in Mitosis and Meiosis

The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.