DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.

DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.     

From early homologue recognition to synaptonemal complex formation

This review focuses on various aspects of chromosome homology searching and their relationship to meiotic and vegetative pairing and to the silencing of unpaired copies of genes. Chromosome recognition and pairing is a prominent characteristic of meiosis; however, for some organisms, this association (complete or partial) is also a normal part of nuclear organization. The multiple mechanisms suggested to contribute to homologous pairing are analyzed. Recognition of DNA/DNA homology also plays an important role in detecting DNA segments that are present in inappropriate number of copies before and during meiosis. In this context, the mechanisms of methylation induced premeiotically, repeat-induced point mutation, meiotic silencing by unpaired DNA, and meiotic sex chromosome inactivation will be discussed. Homologue juxtaposition during meiotic prophase can be divided into three mechanistically distinct steps, namely, recognition, presynaptic alignment, and synapsis by the synaptonemal complex (SC). In most organisms, these three steps are distinguished by their dependence on DNA double-strand breaks (DSBs). The coupling of SC initiation to (and downstream effects of) DSB formation and the exceptions to this dependency are discussed. Finally, this review addresses the specific factors that appear to promote chromosome movement at various stages of meiotic prophase, most particularly at the bouquet stage, and on their significance for homologue pairing and/or achieving a final pachytene configuration.The synaptonemal complex - 50 years     

Direct and Indirect Control of the Initiation of Meiotic Recombination by DNA Damage Checkpoint Mechanisms in Budding Yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.     

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.     

Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form.     

A novel mammalian HORMA domain-containing protein, HORMAD1, preferentially associates with unsynapsed meiotic chromosomes

HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs.     

Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at,or Prior to,Establishment of Double-Strand Breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.     

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.     

A High Throughput Genetic Screen Identifies New Early Meiotic Recombination Functions in Arabidopsis thaliana

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.     

The importance of genetic recombination for fidelity of chromosome pairing in meiosis

In budding yeast, absence of the Hop2 protein leads to extensive synaptonemal complex (SC) formation between nonhomologous chromosomes, suggesting a crucial role for Hop2 in the proper alignment of homologous chromosomes during meiotic prophase. Genetic analysis indicates that Hop2 acts in the same pathway as the Rad51 and Dmc1 proteins, two homologs of E. coli RecA. Thus, the hop2 mutant phenotype demonstrates the importance of the recombination machinery in promoting accurate chromosome pairing. We propose that the Dmc1/Rad51 recombinases require Hop2 to distinguish homologous from nonhomologous sequences during the homology search process. Thus, when Hop2 is absent, interactions between nonhomologous sequences become inappropriately stabilized and can initiate SC formation. Overexpression of RAD51 largely suppresses the meiotic defects of the dmc1 and hop2 mutants. We conclude that Rad51 is capable of carrying out a homology search independently, whereas Dmc1 requires additional factors such as Hop2.     

The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis

Meiosis ensures the reduction of the genome before the formation of generative cells and promotes the exchange of genetic information between homologous chromosomes by recombination. Essential for these events are programmed DNA double strand breaks (DSBs) providing single-stranded DNA overhangs after their processing. These overhangs, together with the RADiation sensitive51 (RAD51) and DMC1 Disrupted Meiotic cDNA1 (DMC1) recombinases, mediate the search for homologous sequences. Current models propose that the two ends flanking a meiotic DSB have different fates during DNA repair, but the molecular details remained elusive. Here we present evidence, obtained in the model plant Arabidopsis thaliana, that the two recombinases, RAD51 and DMC1, localize to opposite sides of a meiotic DSB. We further demonstrate that the ATR kinase is involved in regulating DMC1 deposition at meiotic DSB sites, and that its elimination allows DMC1-mediated meiotic DSB repair even in the absence of RAD51. DMC1's ability to promote interhomolog DSB repair is not a property of the protein itself but the consequence of an ASYNAPTIC1 (Hop1)-mediated impediment for intersister repair. Taken together, these results demonstrate that DMC1 functions independently and spatially separated from RAD51 during meiosis and that ATR is an integral part of the regular meiotic program.     

Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1^-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1^-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1^-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1^-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1^-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.     

A Mec1- and PP4-dependent checkpoint couples centromere pairing to meiotic recombination

The faithful alignment of homologous chromosomes during meiotic prophase requires the coordination of DNA double-strand break (DSB) repair with large-scale chromosome reorganization. Here we identify the phosphatase PP4 (Pph3/Psy2) as a mediator of this process in Saccharomyces cerevisiae. In pp4 mutants, early stages of crossover repair and homology-independent pairing of centromeres are coordinately blocked. We traced the loss of centromere pairing to the persistent phosphorylation of the chromosomal protein Zip1 on serine 75. Zip1-S75 is a consensus site for the ATR-like checkpoint kinase Mec1, and centromere pairing is restored in mec1 mutants. Importantly, Zip1-S75 phosphorylation does not alter chromosome synapsis or DSB repair, indicating that Mec1 separates centromere pairing from the other functions of Zip1. The centromeric localization and persistent activity of PP4 during meiotic prophase suggest a model whereby Zip1-S75 phosphorylation dynamically destabilizes homology-independent centromere pairing in response to recombination initiation, thereby coupling meiotic chromosome dynamics to DSB repair.     

Pds5 is required for homologue pairing and inhibits synapsis of sister chromatids during yeast meiosis

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.     

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.     

Homologous pairing is reduced but not abolished in asynaptic mutants of yeast

In situ hybridization was used to examine chromosome behavior at meiotic prophase in the rad50S, hop1, rad50, and spo11 mutants of Saccharomyces cerevisiae, which are defective in chromosome synapsis and meiotic recombination. Painting of chromosomes I and III revealed that chromosome condensation and pairing are reduced in these mutants. However, there is some residual pairing in meiosis, suggesting that homologue recognition is independent of synaptonemal complex formation and recombination. Association of homologues was observed in the rad50, rad50S, and spo11 mutants, which are defective in the formation or processing of meiotic double-strand breaks. This indicates that double- strand breaks are not an essential component of the meiotic homology searching mechanism or that there exist additional or alternative mechanisms for locating homologues.     

The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair

The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is severely reduced. The Mnd1 protein localizes to chromatin throughout meiotic prophase, and this localization requires Hop2. Unlike recombination enzymes such as Rad51, Mnd1 localizes to chromosomes even in mutants that fail to initiate meiotic recombination. The Hop2 and Mnd1 proteins coimmunoprecipitate from meiotic cell extracts. These results suggest that Hop2 and Mnd1 work as a complex to promote meiotic chromosome pairing and DSB repair. The identification of Hop2 and Mnd1 homologs in other organisms suggests that the function of this complex is conserved among eukaryotes.