Pachytene exit controlled by reversal of Mek1-dependent phosphorylation

During yeast meiosis, a checkpoint prevents exit from pachytene in response to defects in meiotic recombination and chromosome synapsis. This pachytene checkpoint requires two meiotic chromosomal proteins, Red1 and Mek1; Mek1 is a kinase that phosphorylates Red1. In mutants that undergo checkpoint-mediated pachytene arrest, Mek1 is active and Red1 remains phosphorylated. Activation of Mek1 requires the initiation of meiotic recombination and certain DNA damage checkpoint proteins. Mek1 kinase activity and checkpoint-induced pachytene arrest are counteracted by protein phosphatase type 1 (Glc7). Glc7 coimmunoprecipitates with Red1, colocalizes with Red1 on chromosomes, and dephosphorylates Red1 in vitro. We speculate that phosphorylated Red1 prevents exit from pachytene and that completion of meiotic recombination triggers Glc7-dependent dephosphorylation of Red1.     

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.     

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.     

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.     

Genetic requirements and meiotic function of phosphorylation of the yeast axial element protein Red1

The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1(14A) mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.     

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.     

Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic double strand break repair in budding yeast

Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mek1 kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase. We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.     

The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28

Mutants defective in meiotic recombination and synaptonemal complex formation undergo checkpoint-mediated arrest in mid-meiotic prophase. In S. cerevisiae, this checkpoint requires Swe1, which phosphorylates and inactivates the cyclin-dependent kinase Cdc28. A swe1 deletion allows mutants that normally arrest in meiotic prophase to sporulate at wild-type levels, though sporulation is delayed. This delay is eliminated by overproducing Clb1, the major cyclin required for meiosis I. The Swe1 protein accumulates and is hyperphosphorylated in checkpoint-arrested cells. Our results suggest that meiotic arrest is mediated both by increasing Swe1 activity and limiting cyclin production, with Swe1 being the primary downstream target of checkpoint control. The requirement for Swe1 distinguishes the pachytene checkpoint from the DNA damage checkpoints operating in vegetative cells.     

A novel nonnull ZIP1 allele triggers meiotic arrest with synapsed chromosomes in Saccharomyces cerevisiae

During meiotic prophase, assembly of the synaptonemal complex (SC) brings homologous chromosomes into close apposition along their lengths. The Zip1 protein is a major building block of the SC in Saccharomyces cerevisiae. In the absence of Zip1, SC fails to form, cells arrest or delay in meiotic prophase (depending on strain background), and crossing over is reduced. We created a novel allele of ZIP1, zip1-4LA, in which four leucine residues in the central coiled-coil domain have been replaced by alanines. In the zip1-4LA mutant, apparently normal SC assembles with wild-type kinetics; however, crossing over is delayed and decreased compared to wild type. The zip1-4LA mutant undergoes strong checkpoint-induced arrest in meiotic prophase; the defect in cell cycle progression is even more severe than that of the zip1 null mutant. When the zip1-4LA mutation is combined with the pch2 checkpoint mutation, cells sporulate with wild-type efficiency and crossing over occurs at wild-type levels. This result suggests that the zip1-4LA defect in recombination is an indirect consequence of cell cycle arrest. Previous studies have suggested that the Pch2 protein acts in a checkpoint pathway that monitors chromosome synapsis. We hypothesize that the zip1-4LA mutant assembles aberrant SC that triggers the synapsis checkpoint.     

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175 SEP1 arrest at pachytene during meiotic prophase

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175 ^SEP1 enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.     

The Yeast Red1 Protein Localizes to the Cores of Meiotic Chromosomes

Mutants in the meiosis-specific RED1 gene of S. cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC. Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes. Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs. Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC. Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination. The localization of Red1 has been compared with two other meiosisspecific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes. Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene. Double labeling with anti-Hop1and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function. These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact. There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.     

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.     

Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis

Red1, Hop1 and Mek1 are three yeast meiosis-specific chromosomal proteins that uphold the interhomolog (IH) bias of meiotic recombination. Mek1 is also an effector protein kinase in a checkpoint that responds to aberrant DNA and/or axis structure. The activation of Mek1 requires Red1-dependent Hop1-Thr(T)318 phosphorylation, which is mediated by Mec1 and Tel1, the yeast homologs of the mammalian DNA damage sensor kinases ATR and ATM. As the ectopic expression of Mek1-glutathione S-transferase (GST) was shown to promote IH recombination in the absence of Mec1/Tel1-dependent checkpoint function, it was proposed that Mek1 might play dual roles during meiosis by directly phosphorylating targets that are involved in the recombination checkpoint. Here, we report that Mek1 has a positive feedback activity in the stabilization of Mec1/Tel1-mediated Hop1-T318 phosphorylation against the dephosphorylation mediated by protein phosphatase 4. Our results also reveal that GST-Mek1 or Mek1-GST further increases Hop1-T318 phosphorylation. This positive feedback function of Mek1 is independent of Mek1’s kinase activity, but dependent on Mek1’s forkhead-associated (FHA) domain and its arginine 51 residue. Arginine 51 directly mediates the interaction of Mek1-FHA and phosphorylated Hop1-T318. We suggest that the Hop1–Mek1 interaction is similar to the Rad53-Dun1 signaling pathway, which is mediated through the interaction of phosphorylated Rad53 and Dun1-FHA.     

Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein,Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.     

The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation

In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1+ as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.     

Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.     

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.     

Pch2 Prevents Mec1/Tel1-Mediated Hop1 Phosphorylation Occurring Independently of Red1 in Budding Yeast Meiosis

A prominent feature of meiosis in most sexually reproducing organisms is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks are repaired using intact homologous non-sister chromatids rather than sister chromatids. Budding yeast DNA damage checkpoint kinases Mec1 and Tel1 act together with the axial element protein Red1 to promote interhomolog recombination by phosphorylating another axial element protein Hop1. Mec1 and Tel1 also phosphorylate γH2A and the synaptonemal complex protein Zip1 independently of Red1 to facilitate premeiotic DNA replication and to destabilize homology-independent centromere pairing, respectively. It has been unclear why Hop1 phosphorylation is Red1-dependent. Here, we report that the pachytene checkpoint protein 2 (Pch2) specifically prevents Red1-independent Hop1 phosphorylation. Our findings reveal a new function for Pch2 in linking two axial element proteins Red1 and Hop1 thus coordinating their effects in meiotic recombination and the checkpoint network.