Activation of an alternative, rec12 (spo11)-independent pathway of fission yeast meiotic recombination in the absence of a DNA flap endonuclease

Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Delta mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Delta rec12Delta strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.     

Support for a meiotic recombination initiation complex: interactions among Rec102p,Rec104p,and Spo11p

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae requires at least 10 gene products. The initiation event creates double-strand breaks, which are then processed by other recombination enzymes. A variety of classical observations, such as the existence of recombination nodules, have suggested that the proteins catalyzing recombination form a complex. A variety of lines of evidence indicate that Rad50p, Mre11p, and Xrs2p interact, and genetic data suggesting interactions between Rec102p and Rec104p have been reported. It has recently been shown that Spo11p coimmunoprecipitates with Rec102p in meiosis as well. In this paper, we provide genetic and biochemical evidence that the meiosis-specific proteins Rec102p, Rec104p, and Spo11p all interact with each other in meiosis. Furthermore, we demonstrate that the interaction between Rec102p and Spo11p does not require Rec104p. Likewise, the interaction between Rec104p and Rec102p does not require Spo11p, although Spo11p may stabilize that association. The interactions suggest that Spo11p, Rec102p, and Rec104p may form a trimeric complex during the initiation of recombination.     

Sites of strong Rec12/Spo11 binding in the fission yeast genome are associated with meiotic recombination and with centromeres

Meiotic recombination arises from Rec12/Spo11-dependent formation of DNA double-strand breaks (DSBs) and their subsequent repair. We identified Rec12-binding peaks across the Schizosaccharomyces pombe genome using chromatin immunoprecipitation after reversible formaldehyde cross-linking combined with whole-genome DNA microarrays. Strong Rec12 binding coincided with previously identified DSBs at the recombination hotspots ura4A, mbs1, and mbs2 and correlated with DSB formation at a new site. In addition, Rec12 binding corresponded to eight novel conversion hotspots and correlated with crossover density in segments of chromosome I. Notably, Rec12 binding inversely correlated with guanine-cytosine (GC) content, contrary to findings in Saccharomyces cerevisiae. Although both replication origins and Rec12-binding sites preferred AT-rich gene-free regions, they seemed to exclude each other. We also uncovered a connection between binding sites of Rec12 and meiotic cohesin Rec8. Rec12-binding peaks lay often within 2.5 kb of a Rec8-binding peak. Rec12 binding showed preference for large intergenic regions and was found to bind preferentially near to genes expressed strongly in meiosis. Surprisingly, Rec12 binding was also detected in centromeric core regions, which raises the intriguing possibility that Rec12 plays additional roles in meiotic chromosome dynamics.     

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.     

Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination

Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in?a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.     

A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.     

Purification, folding, and characterization of Rec12 (Spo11) meiotic recombinase of fission yeast

Meiotic recombination is initiated by controlled dsDNA breaks (DSBs). Rec12 (Spo11) protein of fission yeast is essential for the formation of meiotic DSBs in vivo, for meiotic recombination, and for segregation of chromosomes during meiosis I. Rec12 is orthologous to Top6A topoisomerase of Archaea and is likely the catalytic subunit of a meiotic recombinase that introduces recombinogenic DSBs. However, despite intensive effort, it has not been possible to produce Rec12 protein in a soluble form required to permit biochemical analyses of function. To obtain purified Rec12 protein for in vitro studies, a rec12(+) cDNA was generated, cloned into vector pET15b(+), and expressed in Escherichia coli. Rec12 protein was produced at moderate levels and it partitioned into insoluble fractions of whole-cell extracts. The protein was enriched based upon its differential solubility in two different denaturants and was further purified by column chromatography. A combinatorial, fractional, factorial approach was used to identify conditions under which Rec12 protein could be refolded. Four parameters were most important and, following optimization, soluble Rec12 protein was obtained. Gel filtration demonstrated that refolded Rec12 protein exists as a monomer in solution, suggesting that additional proteins may be required to assemble biologically-active Rec12 dimers, as inferred previously from genetic data [Cell Chromosome 1 (2002) 1]. The production of refolded Rec12 in a soluble form will allow for characterization in vitro of this key meiotic recombination enzyme.     

Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.     

B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis

BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes. During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation. In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin. Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks. Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process. RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae. We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation. The severity of these phenotypes in each mutant reflects the extent of replication impairment. CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase. Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.     

Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In most sexually reproducing organisms, meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. In budding yeast, nine other proteins are also required for DSB formation, but the roles of these proteins and the interactions among them are poorly understood. We report further studies of the behaviors of these proteins. Consistent with other studies, we find that Mei4 and Rec114 bind to chromosomes from leptonema through early pachynema. Both proteins showed only limited colocalization with the meiotic cohesin subunit Rec8, suggesting that Mei4 and Rec114 associated preferentially with chromatin loops. Rec114 localization was independent of other DSB factors, but Mei4 localization was strongly dependent on Rec114 and Mer2. Systematic deletion analysis identified protein regions important for a previously described two-hybrid interaction between Mei4 and Rec114. We also report functional characterization of a previously misannotated 5′ coding exon of REC102. Sequences encoded in this exon are essential for DSB formation and for Rec102 interaction with Rec104, Spo11, Rec114, and Mei4. Finally, we also examined genetic requirements for a set of previously described two-hybrid interactions that can be detected only when the reporter strain is induced to enter meiosis. This analysis reveals new functional dependencies for interactions among the DSB proteins. Taken together, these studies support the view that Mei4, Rec114, and Mer2 make up a functional subgroup that is distinct from other subgroups of the DSB proteins: Spo11–Ski8, Rec102–Rec104, and Mre11–Rad50–Xrs2. These studies also suggest that an essential function of Rec102 and Rec104 is to connect Mei4 and Rec114 to Spo11. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.     

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.     

Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination

The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.     

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.     

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.     

Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.     

Acetylated Histone H3K9 is associated with meiotic recombination hotspots,and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast.     

A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p.

The Saccharomyces cerevisiae Spo11 protein is thought to catalyze formation of the DNA double-strand breaks that initiate meiotic recombination. We have cloned cDNA and genomic DNA for a mouse gene encoding a protein with significant sequence similarity to conserved domains found in proteins of the Spo11p family. This putative mouse Spo11 gene maps to the distal region of chromosome 2 (homologous to human chromosome 20q13.2-q13.3) and comprises at least 12 exons, spanning approximately 15-18 kb. Strong expression of the Spo11 message is seen in juvenile and adult testis by RNA in situ hybridization, RT-PCR, and Northern blot, with much weaker expression in thymus and brain. In situ hybridization detects expression in oocytes of embryonic ovary, but not of adult ovary. RT-PCR and in situ hybridization analyses of a time course of juvenile testis development indicate that Spo11 expression begins in early meiotic Prophase I, prior to the pachytene stage, with increasing accumulation of mRNA through the pachytene stage. Taken together, these results strongly suggest that this gene encodes the functional homolog of yeast Spo11p, which in turn suggests that the mechanism of meiotic recombination initiation is conserved between yeast and mammals.     

Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.     

Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.