Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over.

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

A new role for the synaptonemal complex in the regulation of meiotic recombination.

Proper segregation during meiosis requires that homologs be connected by the combination of crossovers and sister chromatid cohesion. To generate crossovers, numerous double-strand breaks (DSBs) are introduced throughout the genome by the conserved Spo11 endonuclease. DSB formation and its repair are then highly regulated to ensure that homologous chromosomes contain at least one crossover and no DSBs remain prior to meiosis I segregation. The synaptonemal complex (SC) is a meiosis-specific structure formed between homologous chromosomes during prophase that promotes DSB formation and biases repair of DSBs to homologs over sister chromatids. Synapsis occurs when a particular recombination pathway is successful in establishing stable interhomolog connections. In this issue of Genes & Development, Mu and colleagues (pp. 1605-1618) show that SC formation between individual chromosomes provides the feedback to down-regulate Spo11 activity, thereby revealing an additional function for the SC.

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase.

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed DNA double strand breaks (DSBs). 5' end resection of the DSBs creates 3' single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to involve a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5'-3' helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, and (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates.

Interaction between VPS13A and the XK scramblase is important for VPS13A function in humans.

VPS13 family proteins form conduits between the membranes of different organelles through which lipids are transferred. In humans, there are four VPS13 paralogs, and mutations in the genes encoding each of them are associated with different inherited disorders. VPS13 proteins contain multiple conserved domains. The Vps13 adaptor-binding (VAB) domain binds to adaptor proteins that recruit VPS13 to specific membrane contact sites. This work demonstrates the importance of a different domain in VPS13A function. The pleckstrin homology (PH) domain at the C-terminal region of VPS13A is required to form a complex with the XK scramblase and for the co-localization of VPS13A with XK within the cell. Alphafold modeling was used to predict an interaction surface between VPS13A and XK. Mutations in this region disrupt both complex formation and co-localization of the two proteins. Mutant VPS13A alleles found in patients with VPS13A disease truncate the PH domain. The phenotypic similarities between VPS13A disease and McLeod syndrome caused by mutations in VPS13A and XK, respectively, argue that loss of the VPS13A-XK complex is the basis of both diseases.

Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80.

Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80's function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.

Genetic Dissection of Vps13 Regulation in Yeast Using Disease Mutations from Human Orthologs.

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.

The meiotic-specific Mek1 kinase in budding yeast regulates interhomolog recombination and coordinates meiotic progression with double-strand break repair.

Recombination, along with sister chromatid cohesion, is used during meiosis to physically connect homologous chromosomes so that they can be segregated properly at the first meiotic division. Recombination is initiated by the introduction of programmed double strand breaks (DSBs) into the genome, a subset of which is processed into crossovers. In budding yeast, the regulation of meiotic DSB repair is controlled by a meiosis-specific kinase called Mek1. Mek1 kinase activity promotes recombination between homologs, rather than sister chromatids, as well as the processing of recombination intermediates along a pathway that results in synapsis of homologous chromosomes and the distribution of crossovers throughout the genome. In addition, Mek1 kinase activity provides a readout for the number of DSBs in the cell as part of the meiotic recombination checkpoint. This checkpoint delays entry into the first meiotic division until DSBs have been repaired by inhibiting the activity of the meiosis-specific transcription factor Ndt80, a site-specific DNA binding protein that activates transcription of over 300 target genes. Recent work has shown that Mek1 binds to Ndt80 and phosphorylates it on multiple sites, including the DNA binding domain, thereby preventing Ndt80 from activating transcription. As DSBs are repaired, Mek1 is removed from chromosomes and its activity decreases. Loss of the inhibitory Mek1 phosphates and phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, promote Ndt80 activity such that Ndt80 transcribes its own gene in a positive feedback loop, as well as genes required for the completion of recombination and entry into the meiotic divisions. Mek1 is therefore the key regulator of meiotic recombination in yeast.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Correction: Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

[This corrects the article DOI: 10.1371/journal.pgen.1006226.].

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.

Regulation of meiotic recombination via Mek1-mediated Rad54 phosphorylation.

A preference for homologs over sister chromatids in homologous recombination is a fundamental difference in meiotic versus mitotic cells. In budding yeast, the bias for interhomolog recombination in meiosis requires the Dmc1 recombinase and the meiosis-specific kinase Mek1, which suppresses engagement of sister chromatids by the mitotic recombinase Rad51. Here, a combination of proteomic, biochemical, and genetic approaches has identified an additional role for Mek1 in inhibiting the activity of the Rad51 recombinase through phosphorylation of its binding partner, Rad54. Rad54 phosphorylation of threonine 132 attenuates complex formation with Rad51, and a negative charge at this position reduces Rad51 function in vitro and in vivo. Thus, Mek1 phosphorylation provides a dynamic means of controlling recombination partner choice in meiosis in two ways: (1) it reduces Rad51 activity through inhibition of Rad51/Rad54 complex formation, and (2) it suppresses Rad51-mediated strand invasion of sister chromatids via a Rad54-independent mechanism.

Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks.

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

Recruitment of the lipid kinase Mss4 to the meiotic spindle pole promotes prospore membrane formation in Saccharomyces cerevisiae.

Spore formation in the budding yeast, Saccharomyces cerevisiae, involves de novo creation of four prospore membranes, each of which surrounds a haploid nucleus resulting from meiosis. The meiotic outer plaque (MOP) is a meiosis-specific protein complex associated with each meiosis II spindle pole body (SPB). Vesicle fusion on the MOP surface creates an initial prospore membrane anchored to the SPB. Ady4 is a meiosis-specific MOP component that stabilizes the MOP-prospore membrane interaction. We show that Ady4 recruits the lipid kinase, Mss4, to the MOP. MSS4 overexpression suppresses the ady4∆ spore formation defect, suggesting that a specific lipid environment provided by Mss4 promotes maintenance of prospore membrane attachment to MOPs. The meiosis-specific Spo21 protein is an essential structural MOP component. We show that the Spo21 N terminus contains an amphipathic helix that binds to prospore membranes. A mutant in SPO21 that removes positive charges from this helix shares phenotypic similarities to ady4∆. We propose that Mss4 generates negatively charged lipids in prospore membranes that enhance binding by the positively charged N terminus of Spo21, thereby providing a mechanism by which the MOP-prospore membrane interaction is stabilized.

Mek1 kinase is regulated to suppress double-strand break repair between sister chromatids during budding yeast meiosis.

Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1Delta-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpoint-induced arrest.

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1.

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

DNA Helicase Mph1FANCM Ensures Meiotic Recombination between Parental Chromosomes by Dissociating Precocious Displacement Loops.

Meiotic pairing between parental chromosomes (homologs) is required for formation of haploid gametes. Homolog pairing depends on recombination initiation via programmed double-strand breaks (DSBs). Although DSBs appear prior to pairing, the homolog, rather than the sister chromatid, is used as repair partner for crossing over. Here, we show that Mph1, the budding yeast ortholog of Fanconi anemia helicase FANCM, prevents precocious DSB strand exchange between sister chromatids before homologs have completed pairing. By dissociating precocious DNA displacement loops (D-loops) between sister chromatids, Mph1FANCM ensures high levels of crossovers and non-crossovers between homologs. Later-occurring recombination events are protected from Mph1-mediated dissociation by synapsis protein Zip1. Increased intersister repair in absence of Mph1 triggers a shift among remaining interhomolog events from non-crossovers to crossover-specific strand exchange, explaining Mph1's apparent anti-crossover function. Our findings identify temporal coordination between DSB strand exchange and homolog pairing as a critical determinant for recombination outcome.

Persistent DNA-break potential near telomeres increases initiation of meiotic recombination on short chromosomes.

Faithful meiotic chromosome inheritance and fertility rely on the stimulation of meiotic crossover recombination by potentially genotoxic DNA double-strand breaks (DSBs). To avoid excessive damage, feedback mechanisms down-regulate DSBs, likely in response to initiation of crossover repair. In Saccharomyces cerevisiae, this regulation requires the removal of the conserved DSB-promoting protein Hop1/HORMAD during chromosome synapsis. Here, we identify privileged end-adjacent regions (EARs) spanning roughly 100 kb near all telomeres that escape DSB down-regulation. These regions retain Hop1 and continue to break in pachynema despite normal synaptonemal complex deposition. Differential retention of Hop1 requires the disassemblase Pch2/TRIP13, which preferentially removes Hop1 from telomere-distant sequences, and is modulated by the histone deacetylase Sir2 and the nucleoporin Nup2. Importantly, the uniform size of EARs among chromosomes contributes to disproportionately high DSB and repair signals on short chromosomes in pachynema, suggesting that EARs partially underlie the curiously high recombination rate of short chromosomes.

Partner choice during meiosis is regulated by Hop1-promoted dimerization of Mek1.

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.

Chemical inactivation of cdc7 kinase in budding yeast results in a reversible arrest that allows efficient cell synchronization prior to meiotic recombination.

Genetic studies in budding yeast have provided many fundamental insights into the specialized cell division of meiosis, including the identification of evolutionarily conserved meiosis-specific genes and an understanding of the molecular basis for recombination. Biochemical studies have lagged behind, however, due to the difficulty in obtaining highly synchronized populations of yeast cells. A chemical genetic approach was used to create a novel conditional allele of the highly conserved protein kinase Cdc7 (cdc7-as3) that enables cells to be synchronized immediately prior to recombination. When Cdc7-as3 is inactivated by addition of inhibitor to sporulation medium, cells undergo a delayed premeiotic S phase, then arrest in prophase before double-strand break (DSB) formation. The arrest is easily reversed by removal of the inhibitor, after which cells rapidly and synchronously proceed through recombination and meiosis I. Using the synchrony resulting from the cdc7-as3 system, DSB-dependent phosphorylation of the meiosis-specific chromosomal core protein, Hop1, was shown to occur after DSBs. The cdc7-as3 mutant therefore provides a valuable tool not only for understanding the role of Cdc7 in meiosis, but also for facilitating biochemical and cytological studies of recombination.