Morphogenesis of the synapton during yeast meiosis

The formation of the synapton (synaptonemal complex) was followed by an electron microscopic examination of large samples of Saccharomyces cerevisiae cells at various stages of meiosis. Three temperature-sensitive mutants were used, cdc4, cdc5 and cdc7, which undergo a slow but normal meiosis at 25° C. At the restrictive temperature of 34° C, cdc4 and cdc5 arrest at an advanced enough stage of meiosis to allow the study of synapton morphogenesis. Based on the frequencies of nuclear structures, we describe the formation of the central region and central elements of the synapton in the dense body, which may be part of the nucleolus. This process occurs during early meiotic stages, concomittantly with recombination commitment and premeiotic DNA replication. Mature synaptons usually appear after premeiotic S, at the pachytene stage, and later disappear. A possible intermediate stage in this disappearance is found in arrested cdc5 cells, which contain paired lateral elements without central elements. — Following the frequencies of spindle plaque configurations, we conclude that the plaques in meiosis duplicate once at the beginning of the main DNA replication, as is also observed prior to mitosis. In contrast to mitotic cells, however, meiotic plaques remain duplicated for a long period, until the synaptons disappear, and only then separate from each other to form a spindle. During late stages of the first meiotic division, the outer plates of the spindle plaques thicken, to duplicate later and give the second division spindles. The characteristically thick outer plate may have a role in the formations of the ascospore wall.     

The effect of the cdc9 mutation on premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

A diploid homozygous for cdc9, a conditional mutation defective in DNA ligase [2], has been used to investigate the role of this enzyme in premeiotic DNA synthesis. The cdc9 ligase has the same effect on premeiotic as on mitotic DNA synthesis and at the restrictive temperature the newly synthesized DNA is recovered in small fragments. A difference has been observed, however, between meiotic and mitotic cells, namely in their ability to join together these fragments on return to the permissive temperature. In mitotic cells this can be readly demonstrated within 50 min, whereas in contrast little joining was detected in meiotic cells, even after 2 h at the permissive temperature.     

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 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.     

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).     

Meiosis in a temperature-sensitive DNA-synthesis mutant and in an apomictic yeast strain (Saccharomyces cerevisiae).

It is shown that in the temperature-sensitive yeast mutant (Saccharomyces cerevisiae) spo 11 at the restrictive temperature of 34 degrees C. (1) premeiotic DNA synthesis is nearly completely blocked; (2) the nucleus enters meiotic prophase indicated by the formation of axial cores and polysynaptonemal complexes; (3) the kinetic apparatus functions normally at meiosis I and II; (4) early spore formation occurs in nearly all cells but it is variable and all spores eventually degenerate. It is concluded that chromosome replication is not a prerequisite for the functions listed above. The apomictic yeast strain 4117 produces 2 diploid spores. It is shown that a diploid which produces 2-spored asci, synthesized from 4117, no. 5, and an adenine requiring strain (1) has a normal meiotic prophase with abundant synaptonemal complexes; (2) has only one meiotic spindle; (3) has spores which form red clones more frequently than normal or u.v.-treated vegetative cells form ade/ade red sectors through mitotic recombination. It is concluded that this apomictic yeast has maintained meiotic prophase, but that one of the two meiotic divisions is suppressed.     

Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature

Summary The temperature sensitivity of sporulation in a well-characterized yeast strain lacking any known temperature sensitive genes has been investigated. Cytological observations by electron microscopy demonstrate that cells incubated in sporulation medium at a temperature inhibitory to sporulation became arrested in meiotic prophase. The stage of arrest was identified as pachytene by the presence of duplicated (but unseparated) spindle pole bodies and synaptonemal complex. Transfer of the arrested culture to lower temperature permitted resumption of meiosis and sporulation; transfer to vegetative medium resulted in reversion to mitotic division. Genetic analysis of cells that had reverted to mitosis revealed that commitment to intragenic recombination had occurred by the time of arrest. Prolonged incubation at the elevated temperature resulted in the enhancement of intragenic recombination above normal levels, suggesting that some aspect of recombination continued to occur during the pachytene arrest. Evidence is presented that DNA replication, although depressed overall in the arrested cultures, had occurred to completion in many arrested cells.     

PP2ACdc55 is required for multiple events during meiosis I

Protein phosphatase 2A (PP2A) is a heterotrimer consisting of A and B regulatory subunits and a C catalytic subunit. PP2A regulates mitotic cell events that include the cell cycle, nutrient sensing, p53 stability and various mitogenic signals. The role of PP2A during meiosis is less understood. We explored the role of Saccharomyces cerevisiae PP2A during meiosis. We show a PP2A^Cdc55 containing the human B/55 family B subunit ortholog, Cdc55, is required for progression through meiosis I. Mutant cells lacking Cdc55 remain mononucleated. They harbor meiotic gene expression, premeiotic DNA replication, homologous recombination and spindle pole body (SPB) defects. They initiate but do not complete replication and are defective in performing intergenic homologous recombination. Bypass alleles, which allow cells defective in recombination to finish meiosis, do not suppress the meiosis I defect. cdc55 cells arrest with a single SPB lacking microtubules, or duplicated but not separated SBPs containing microtubules. Finally, the premeiotic replication defect is suppressed by loss of Rad9 checkpoint function. We conclude PP2A^Cdc55 is required for the proper temporal initiation of multiple meiotic events and/or monitors these events to ensure their fidelity.     

Meiotic Recombination and DNA Synthesis in a New Cell Cycle Mutant of SACCHAROMYCES CEREVISIAE

Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.     

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.     

Pds1p Is Required for Meiotic Recombination and Prophase I Progression in Saccharomyces cerevisiae

Sister-chromatid separation at the metaphase–anaphase transition is regulated by a proteolytic cascade. Destruction of the securin Pds1p liberates the Esp1p separase, which ultimately targets the mitotic cohesin Mcd1p/Scc1p for destruction. Pds1p stabilization by the spindle or DNA damage checkpoints prevents sister-chromatid separation while mutants lacking PDS1 (pds1Δ) are temperature sensitive for growth due to elevated chromosome loss. This report examined the role of the budding yeast Pds1p in meiotic progression using genetic, cytological, and biochemical assays. Similar to its mitotic function, Pds1p destruction is required for metaphase I–anaphase I transition. However, even at the permissive temperature for growth, pds1Δ mutants arrest with prophase I spindle and nuclear characteristics. This arrest was partially suppressed by preventing recombination initiation or by inactivating a subset of recombination checkpoint components. Further studies revealed that Pds1p is required for recombination in both double-strand-break formation and synaptonemal complex assembly. Although deleting PDS1 did not affect the degradation of the meiotic cohesin Rec8p, Mcd1p was precociously destroyed as cells entered the meiotic program. This role is meiosis specific as Mcd1p destruction is not altered in vegetative pds1Δ cultures. These results define a previously undescribed role for Pds1p in cohesin maintenance, recombination, and meiotic progression.     

Meiotic recombination and synaptonemal complexes in Saccharomyces cerevisiae.

Summary The course of meiotic recombination, gene conversion and crossing-over, was investigated in Saccharomyces cerevisiae. Gene conversion was used as the selected event by removing cells from a medium inducing and promoting meiosis to a vegetative growth medium selective for convertants. Gene conversion started to increase at the same time as DNA synthesis, and nuclei entered a phase where the chromatin appeared as thread-like structures. Crossing-over of linked and unlinked markers also started early but remained at a low level until synaptonemal complexes were formed. However, gene conversion and a limited amount of crossing-over could be completed without synaptonemal complexes. It was concluded that meiotic recombination in yeast can occur as early as during DNA synthesis and does not require the function of synaptonemal complexes. Moreover, the low incidence of crossing-over early in meiosis is attributed to a low frequency of strand isomerization.     

Elevated recombination and pairing structures during meiotic arrest in yeast of the nuclear division mutant cdc5

Summary A diploid strain of yeast, homozygous for the mutation cdc5-1, undergoes a normal meiosis at 25° C. At the nonpermissive temperature of 34° C, meiosis is arrested at the first meiotic division, after premeiotic DNA replication and recombination commitment have taken place. Haploidisation commitment does not occur at 34° C. Electron microscopy reveals that synaptons (synaptonemal complexes) are formed and the stage of arrest is characterised by a prevalence of modified synaptons, which consist of paired lateral elements lacking the central elements. Prolonged incubation at this stage of arrest results in unusually high recombination levels, perhaps related to the synaptonal structures observed.Temperature shift-up experiments (transfers of cells from 25° C to 34° C at various times during meiosis) reveal that the CDC5 function is required for both the first and the second divisions of meiosis.     

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.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.     

Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II

To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mitotically to disentangle sister chromatids at the time of chromosome segregation. In meiosis, we found that topoisomerase II is required only at the time of nuclear division. When cold-sensitive top2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division. The introduction of a mutation blocking recombination relieves the requirement for topoisomerase II in meiosis I. These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes.     

A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis

 In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation. Received: 20 February 1996 / Accepted: 7 June 1996     

Meiosis-specific arrest revealed in DNA topoisomerase II mutants.

Although the processes of mitosis and meiosis are similar, there is evidence for fundamental regulatory differences between the two. To examine these differences, we have compared the meiotic phenotype of DNA topoisomerase II mutants with their previously described mitotic phenotype (C. Holm, T. Goto, J. Wang, and D. Botstein, Cell 41:553-563, 1985). top2 mutants in meiosis show no defects in the latest detectable stages of recombination, yet they arrest prior to spindle establishment at meiosis I. Fluorescence and electron microscopy reveal that top2 mutants exhibit wild-type levels of meiotic chromosome condensation and form morphologically normal synaptonemal complex but are delayed in the exit from pachytene. Arrested cells retain viability and form colonies if transferred to mitotic medium. Our results suggest that the top2 meiotic arrest is regulatory in nature. This arrest may have evolved to ensure the resolution of fortuitous tangles between nonhomologous chromosomes.     

The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.