The budding yeast Msh4 protein functions in chromosome synapsis and the regulation of crossover distribution.

The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.     

A Role for SUMO in meiotic chromosome synapsis

During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. Here, we have investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. We show that SUMO localizes specifically to synapsed regions of meiotic chromosomes and that this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. Our data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis.     

Full-Length Synaptonemal Complex Grows Continuously during Meiotic Prophase in Budding Yeast

The synaptonemal complex (SC) links two meiotic prophase chromosomal events: homolog pairing and crossover recombination. SC formation involves the multimeric assembly of coiled-coil proteins (Zip1 in budding yeast) at the interface of aligned homologous chromosomes. However, SC assembly is indifferent to homology and thus is normally regulated such that it occurs only subsequent to homology recognition. Assembled SC structurally interfaces with and influences the level and distribution of interhomolog crossover recombination events. Despite its involvement in dynamic chromosome behaviors such as homolog pairing and recombination, the extent to which SC, once installed, acts as an irreversible tether or maintains the capacity to remodel is not clear. Experiments presented here reveal insight into the dynamics of the full-length SC in budding yeast meiotic cells. We demonstrate that Zip1 continually incorporates into previously assembled synaptonemal complex during meiotic prophase. Moreover, post-synapsis Zip1 incorporation is sufficient to rescue the sporulation defect triggered by SCs built with a mutant version of Zip1, Zip1-4LA. Post-synapsis Zip1 incorporation occurs initially with a non-uniform spatial distribution, predominantly associated with Zip3, a component of the synapsis initiation complex that is presumed to mark a subset of crossover sites. A non-uniform dynamic architecture of the SC is observed independently of (i) synapsis initiation components, (ii) the Pch2 and Pph3 proteins that have been linked to Zip1 regulation, and (iii) the presence of a homolog. Finally, the rate of SC assembly and SC central region size increase in proportion to Zip1 copy number; this and other observations suggest that Zip1 does not exit the SC structure to the same extent that it enters. Our observations suggest that, after full-length assembly, SC central region exhibits little global turnover but maintains differential assembly dynamics at sites whose distribution is patterned by a recombination landscape.     

Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins

The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase. Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing. Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes. These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene.     

Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis

Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the “ZMM” genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.     

Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice.

Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.     

Three Distinct Modes of Mec1/ATR and Tel1/ATM Activation Illustrate Differential Checkpoint Targeting during Budding Yeast Early Meiosis

Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.     

Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of sgs1

Sgs1, the budding yeast homolog of the mammalian BLM helicase, has been implicated in preventing excess recombination during both vegetative growth and meiosis. Most meiotic crossover (CO) recombination requires full function of a set of yeast proteins (Zip1, Zip2, Zip3, Zip4/Spo22, Mer3, Msh4, and Msh5, termed the SIC or ZMM proteins) that are also required for homologous chromosome synapsis. We report here genetic and molecular assays showing that sgs1 single mutants display relatively modest increases in CO recombination (less than 1.6-fold relative to wild-type). In contrast, a much greater CO increase is seen when an sgs1 mutation is introduced into the CO- and synapsis-deficient zip1, zip2, zip3, mer3, or msh4 mutants (2- to 8-fold increase). Furthermore, close juxtaposition of the axes of homologous chromosomes is restored. CO restoration in the mutants is not accompanied by significant changes in noncrossover (NCO) recombinant frequencies. These findings show that Sgs1 has potent meiotic anti-CO activity, which is normally antagonized by SIC/ZMM proteins. Our data reinforce previous proposals for an early separation of meiotic processes that form CO and NCO recombinants.     

Pch2 links chromatin silencing to meiotic checkpoint control.

The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective. Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability. Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein. Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest. Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function. These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint.     

Separation of roles of Zip1 in meiosis revealed in heterozygous mutants of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Synapsis of homologs during meiotic prophase I is associated with a protein complex built along the bivalents—the synaptonemal complex (SC). Mutations in the SC-component gene ZIP1 diminish SC formation, leading to reduced recombination levels and low spore viability. Here we show that in SK1 strains heterozygous for a deletion of ZIP1 in certain regions meiotic interference are impaired with no decrease in recombination levels. The extent of synapsis is over all reduced and NDJ levels of a large endogenous chromosome and of artificial chromosomes (YACs) rise to twice the level of wild type strains. A substantial proportion of mis-segregating YACs had undergone crossing over. This demonstrates that different functions of Zip1 display differential sensitivities to changes in expression levels.     

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.     

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.     

The meiosis-specific zip4 protein regulates crossover distribution by promoting synaptonemal complex formation together with zip2

We have characterized Zip4 (a.k.a. Spo22), a meiosis-specific protein essential for chromosome synapsis in budding yeast. In the absence of Zip4, the synaptonemal complex protein Zip1 fails to polymerize along chromosomes. Zip2 and Zip3 are previously characterized components of the synapsis initiation complex. Zip4 forms a functional unit with Zip2 that is distinct from Zip3. Zip2 and Zip4 are mutually dependent for their chromosomal localization; in polycomplexes, the pattern of Zip2/Zip4 localization is distinct from that of Zip3. Crossing-over is decreased in the zip4 mutant (as in zip1, zip2, and zip3); the remaining crossovers are largely dependent on a parallel pathway utilizing Mms4. zip4 displays a novel phenotype: negative crossover interference, meaning that crossovers tend to cluster. This clustering depends on Zip1. Our results suggest an interaction between crossover pathways such that a protein (Zip1) acting in one pathway influences the distribution of crossovers promoted by a parallel (Mms4-dependent) pathway.     

Analysis of the kar3 meiotic arrest in Saccharomyces cerevisiae

The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint.     

Analysis of the kar3 Meiotic Arrest in Saccharomyces cerevisiae

The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint.     

Disruption of pairing and synapsis of chromosomes causes stage-specific apoptosis of male meiotic cells

During meiosis, DNA replication is followed by two successive rounds of chromosome segregation (meiosis I and II), which give rise to genetically diverse haploid gametes. The prophase of the first meiotic division is highly regulated and alignment and synapsis of the homologous chromosomes during this stage are mediated by the synaptonemal complex. Incorrect assembly of the synaptonemal complex results in cell death, impaired meiotic recombination and aneuploidy. Oocytes with meiotic defects often survive the first meiotic prophase and give rise to aneuploid gametes. Similarly affected spermatocytes, on the other hand, almost always undergo apoptosis at a male-specific meiotic checkpoint, located specifically at epithelial stage IV during spermatogenesis. Many examples of this stage IV-specific arrest have been described for several genetic mouse models in which DNA repair or meiotic recombination are abrogated. Interestingly, in C. elegans, meiotic recombination and synapsis are monitored by two separate checkpoint pathways. Therefore we studied spermatogenesis in several knockout mice (Sycp1(-/-), Sycp3(-/-), Smc1beta(-/-) and Sycp3/Sycp1 and Sycp3/Smc1beta double-knockouts) that are specifically defective in meiotic pairing and synapsis. Like for recombination defects, we found that all these genotypes also specifically arrest at epithelial stage IV. It seems that the epithelial stage IV checkpoint eliminates spermatocytes that fail a certain quality check, being either synapsis or DNA damage related.     

Saccharomyces cerevisiae Mer3 is a DNA helicase involved in meiotic crossing over

Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.     

Role for the silencing protein Dot1 in meiotic checkpoint control

During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.