A Rad9-Dependent Checkpoint Blocks Meiosis of Cdc13 Yeast Cells

Mutations in CDC13 have previously been found to cause cell cycle arrest of Saccharomyces cerevisiae at a stage in G2 immediately preceding the mitotic division. We show here that cdc13 blocks the meiotic pathway at a stage that follows DNA replication, but in this case the spindle has not yet formed nor have the chromosomes undergone synapsis or recombination. This arrest is alleviated by rad9, thus implicating the same checkpoint function that delays mitotic progression when chromosomal lesions are present. An assessment of the spores produced upon alleviation of the meiotic arrest by rad9 reveals that the absence of recombination in strains bearing cdc13 alone is attributable to the RAD9-mediated arrest rather than to other effects of cdc13 lesions. We have tested the possibility that this checkpoint function is important in regulating meiotic progression to permit resolution of recombinational intermediates during ongoing meiosis and have found no evidence that rad9 alters the execution of functions that might depend upon such regulation. We consider the possible role of other checkpoints in yeast meiosis.     

Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the double-strand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G2, these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

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.     

Me14, a Yeast Gene Required for Meiotic Recombination

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.     

Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange.     

Relationships between a Hyper-Rec Mutation (rem1 ) and Other Recombination and Repair Genes in Yeast

Mutations in the REM1 gene of Saccharomyces cerevisiae confer a semidominant hyper-recombination and hypermutable phenotype upon mitotic cells ( GOLIN and ESPOSITO 1977). These effects have not been observed in meiosis. We have examined the interactions of rem1 mutations with rad6-1, rad50 -1, rad52-1 or spo11 -1 mutations in order to understand the basis of the rem1 hyper-rec phenotype. The rad mutations have pleiotropic phenotypes; spo11 is only defective in sporulation and meiosis. The RAD6, RAD50 and SPO11 genes are not required for spontaneous mitotic recombination; mutations in the RAD52 gene cause a general spontaneous mitotic Rec- phenotype. Mutations in RAD50 , RAD52 or SPO11 eliminate meiotic recombination, and mutations in RAD6 prevent spore formation. Evidence for the involvement of RAD6 in meiotic recombination is less clear. Mutations in all three RAD genes confer sensitivity to X rays; the RAD6 gene is also required for UV damage repair. To test whether any of these functions might be involved in the hyper-rec phenotype conferred by rem1 mutations, double mutants were constructed. Double mutants of rem1 spo11 were viable and demonstrated rem1 levels of mitotic recombination, suggesting that the normal meiotic recombination system is not involved in producing the rem1 phenotype. The rem1 rad6 double mutant was also viable and had rem1 levels of mitotic recombination. Neither rem1 rad50 nor rem1 rad52 double mutants were viable. This suggests that rem1 causes its hyper-rec phenotype because it creates lesions in the DNA that are repaired using a recombination-repair system involving RAD50 and RAD52.     

Effects of the Mitotic Cell-Cycle Mutation cdc4 on Yeast Meiosis

The mitotic cell-cycle mutation cdc4 has been reported to block the initiation of nuclear DNA replication and the separation of spindle plaques after their replication. Meiosis in cdc4/cdc4 diploids is normal at the permissive temperature (25 degrees) and is arrested at the first division (one-nucleus stage) at the restrictive temperature (34 degrees or 36 degrees). Arrested cells at 34 degrees show a high degree of commitment to recombination (at least 50% of the controls) but no haploidization, while cells arrested at 36 degrees are not committed to recombination. Meiotic cells arrested at 34 degrees show a delayed and reduced synthesis of DNA (at most 40% of the control), at least half of which is probably mitochondrial. It is suggested that recombination commitment does not depend on the completion of nuclear premeiotic DNA replication in sporulation medium.--Transfer of cdc4/cdc4 cells to the restrictive temperature at the onset of sporulation produces a uniform phenotype of arrest at a 1-nucleus morphology. On the other hand, shifts of the meiotic cells to the restrictive temperature at later times produce two additional phenotypes of arrest, thus suggesting that the function of cdc4 is required at several points in meiosis (at least at three different times).     

The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.     

Meiotic S-phase damage activates recombination without checkpoint arrest

Checkpoints operate during meiosis to ensure the completion of DNA synthesis and programmed recombination before the initiation of meiotic divisions. Studies in the fission yeast Schizosaccharomyces pombe suggest that the meiotic response to DNA damage due to a failed replication checkpoint response differs substantially from the vegetative response, and may be influenced by the presence of homologous chromosomes. The checkpoint responses to DNA damage during fission yeast meiosis are not well characterized. Here we report that DNA damage induced during meiotic S-phase does not activate checkpoint arrest. We also find that in wild-type cells, markers for DNA breaks can persist at least to the first meiotic division. We also observe increased spontaneous S-phase damage in checkpoint mutants, which is repaired by recombination without activating checkpoint arrest. Our results suggest that fission yeast meiosis is exceptionally tolerant of DNA damage, and that some forms of spontaneous S-phase damage can be repaired by recombination without activating checkpoint arrest.     

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.     

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.     

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.     

Meiotic Recombination and Sporulation in Repair-Deficient Strains of Yeast

A genetic system designed to monitor recombination and sporulation in various repair-deficient yeast strains was constructed. Variously heterozygous at seven or eight sites distributed across the genome, the system facilitated sensitive detection of changes in frequency or pattern of meiotic recombination. Ten rad mutants sensitive primarily to UV-irradiation and without terminal blocks in the sporulation process were studied. Seven were defective in excision repair (rad1, rad2, rad3, rad4, rad10, rad14 and rad16), and three were defective in mutagenic repair (rad5, rad9 and rad18). Individually, each mutant displayed behavior consistent with an orthodox meiosis including a wild-type meiotic recombination profile with respect to gene conversion, PMS and intergenic map distances. Accordingly, we conclude that these mutants are without major effect on meiotic heteroduplex formation or correction. However, certain combinations of excision-defective mutants with rad18 exhibited marked ascosporal inviability. Tetraploids homozygous for rad1 and rad18 produce a large proportion of diploid spores containing a recessive lethal.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Molecular and Genetic Analysis of Rec103, an Early Meiotic Recombination Gene in Yeast

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, MEI4, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SKI8, a gene that depresses the expression of yeast double-stranded (``killer') (ds)RNA viruses. REC103/SKI8 is transcribed in mitotic cells and is induced ~15-fold in meiosis. REC103 has 26% amino acid identity to the Schizosaccharomyces pombe rec14(+) gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.     

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.