Premeiotic S-phase Research Articles

FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination

The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs essential for homology search and strand exchange in DNA double-strand break (DSB) repair is tightly regulated. FIGNL1 AAA+++ ATPase controls RAD51-mediated recombination in human cells. However, its role in gametogenesis remains unsolved. Here, we characterized a germ line-specific conditional knockout (cKO) mouse of FIGNL1. Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on meiotic chromosomes, supporting a positive role of FIGNL1 in homologous recombination at a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes also accumulate RAD51/DMC1 on chromosomes in pre-meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. We also showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest an additional role of FIGNL1 in limiting the non-productive assembly of RAD51/DMC1 on native dsDNAs during pre-meiotic S-phase and meiotic prophase I.

Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Vir1p, the yeast homolog of virilizer, is required for mRNA m6A methylation and meiosis.

N6-Methyladenosine (m6A) is among the most abundant modifications of eukaryotic mRNAs. mRNA methylation regulates many biological processes including playing an essential role in meiosis. During meiosis in the budding yeast, Saccharomyces cerevisiae, m6A levels peak early, before the initiation of the meiotic divisions. High-throughput studies suggested, and this work confirms that the uncharacterized protein Ygl036wp interacts with Kar4p, a component of the mRNA m6A-methyltransferase complex. Protein structure programs predict that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m6A-methylation in higher eukaryotes. In addition, Ygl036wp contains conserved motifs shared with VIRMA/Virilizer/VIR. Accordingly, we propose the name VIR1 for budding yeast ortholog of VIRMA/Virilizer/VIR 1. Vir1p interacts with all other members of the yeast methyltransferase complex and is itself required for mRNA m6A methylation and meiosis. In the absence of Vir1p proteins comprising the methyltransferase complex become unstable, suggesting that Vir1p acts as a scaffold for the complex. The vir1Δ/Δ mutant is defective for the premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. The vir1Δ/Δ mutant exhibits reduced levels of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed an additional defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by overexpression of RIM4. Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation.

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

Therapeutic Dose of Hydroxyurea-Induced Synaptic Abnormalities on the Mouse Spermatocyte.

Hydroxyurea (HU) is a widely used pharmacological therapy for sickle cell disease (SCD). However, replication stress caused by HU has been shown to inhibit premeiotic S-phase DNA, leading to reproductive toxicity in germ cells. In this study, we administered the therapeutic doses of HU (i.e., 25 and 50 mg/kg) to male mice to explore whether replication stress by HU affects pachytene spermatocytes and causes the abnormalities of homologous chromosomes pairing and recombination during prophase I of meiosis. In comparison with the control group, the proportions of spermatocyte gaps were significantly different in the experimental groups injected with 25 mg/kg (p < 0.05) and 50 mg/kg of HU (p < 0.05). Moreover, the proportions of unrepaired double-stranded breaks (DSBs) observed by γH2AX staining also corresponded to a higher HU dose with a greater number of breaks. Additionally, a reduction in the counts of recombination foci on the autosomal SCs was observed in the pachytene spermatocytes. Our results reveal that HU has some effects on synaptonemal complex (SC) formation and DSB repair which suggest possible problems in fertility. Therefore, this study provides new evidence of the mechanisms underlying HU reproductive toxicity.

Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

Mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe.

The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a ‘sponge RNA’ for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2. There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2–meiRNA a ‘double sponge’ (i.e. binding both an mRNA and its encoded protein).

Direct Evidence for Sister Kinetochore Fusion in Meiosis I

For decades, biologists have wondered how replicated sister chromatids, which always separate from one another during normal mitotic cell division, instead co-migrate together during phase I of meiosis, the specialized division that produces eggs and sperm. Genetic, cytological, and biochemical studies using budding yeast have suggested that sister kinetochores, which link the sister chromatids to microtubules and drive their movement, may be mechanically fused by a meiosis I-specific kinetochore-binding factor called monopolin. But thus far the evidence for monopolin-dependent sister kinetochore fusion has been indirect, and the possibility that monopolin could instead promote co-migration by binding to and inhibiting one of the two sister kinetochores has not been excluded. To test these ideas directly, we are isolating native meiotic and mitotic kinetochore particles, reconstituting their function in vitro, and applying advanced tools for manipulating and tracking individual molecules. Using laser trapping, we find that kinetochore particles from meiosis I form substantially stronger attachments to dynamic microtubule tips than those from mitosis or from meiosis II. The high strength of meiosis I particles is lost if DNA replication is blocked during pre-meiotic S-phase, demonstrating that sisters are required. High strength is also lost if monopolin is disrupted prior to kinetochore particle isolation and, conversely, the strength of mitotic kinetochores can be increased by artificially inducing monopolin expression. Quantitative fluorescence microscopy also confirms that meiosis I particles have more copies of the core microtubule-binding component, Nuf2. Together these data suggest that sister kinetochores are mechanically fused by the monopolin complex during meiosis I, in such a way that the microtubule-binding elements from the two sister kinetochores can cooperate together to form a single attachment site.

Biophysical Measurements Reveal Fusion of Sister Kinetochores during Meiosis I

Sister chromatids move toward opposite poles of the spindle during mitosis. Strikingly however, they must instead co-migrate toward the same pole during phase I of meiosis, the specialized cell division that produces sperm and eggs. Sister co-migration in budding yeast requires a meiosis I-specific protein complex called monopolin that possibly acts by mechanically fusing sister kinetochores (on the corresponding sister chromatids). Since sister kinetochore fusion might bring together twice as many microtubule-binding elements, we hypothesized that meiosis I kinetochores could form stronger attachments to microtubules than mitotic or meiosis II kinetochores. To test this idea we purified kinetochore particles from meiotic or mitotic budding yeast, attached them to individual dynamic microtubules in vitro, and then measured their rupture strength at the single particle level using a computer-controlled laser trap operating in force-ramp mode. Consistent with the fusion model, meiosis I kinetochores ruptured at 13.09 ± 0.47 pN (mean ± s.e.m) on average, significantly higher than the strengths of mitotic and meiosis II kinetochores, 9.61 ± 0.56 pN and 9.30 ± 0.73 pN, respectively. Blocking DNA replication during pre-meiotic S-phase reduced the high strength of meiosis I kinetochores, making them rupture at mitotic-like levels, 9.31 ± 1.22 pN, confirming that sister chromatid's presence is essential. High strength of meiosis I kinetochores was also lost when monopolin was disrupted and conversely, strength of mitotic kinetochores upon ectopic expression of monopolin increased to 11.28 ± 0.90 pN, an intermediate value suggesting a mix of fused sisters and individual, un-fused kinetochores. Addition of recombinant monopolin to mitotic kinetochores in vitro was also sufficient to increase their strength to meiosis I-like levels, 13.00 ± 0.86 pN. Taken together, these data provide strong direct evidence for a monopolin-dependent mechanical fusion of sister kinetochores during meiosis I.

Ipl1/Aurora Kinase Suppresses S-CDK-Driven Spindle Formation during Prophase I to Ensure Chromosome Integrity during Meiosis

Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

A Novel RNA-Recognition-Motif Protein Is Required for Premeiotic G1/S-Phase Transition in Rice (Oryza sativa L.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.

PCH'ing Together an Understanding of Crossover Control

PCH'ing Together an Understanding of Crossover Control

Differences in DNA double strand breaks repair in male germ cell types: Lessons learned from a differential expression of Mdc1 and 53BP1

In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines. Irradiation induced immunofluorescent foci (IRIF's) signifying a double strand DNA breaks, were followed in spermatogenic cells up to 16 h after the insult. Foci were characterised for Mdc1, 53BP1 and Rad51 that always were expressed in conjecture with γ-H2AX. Subsequent spermatogenic cell types were found to have different repair proteins. In early germ cells up to the start of meiotic prophase, i.e. in spermatogonia and preleptotene spermatocytes, 53BP1 and Rad51 are available but no Mdc1 is expressed in these cells before and after irradiation. The latter might explain the radiosensitivity of spermatogonia. Spermatocytes from shortly after premeiotic S-phase till pachytene in epithelial stage IV/V express Mdc1 and Rad51 but no 53BP1 which has no role in recombination involved repair during the early meiotic prophase. Mdc1 is required during this period as in Mdc1 deficient mice all spermatocytes enter apoptosis in epithelial stage IV when they should start mid-pachytene phase of the meiotic prophase. From stage IV mid pachytene spermatocytes to round spermatids, Mdc1 and 53BP1 are expressed while Rad51 is no longer expressed in the haploid round spermatids. Quantifying foci numbers of γ-H2AX, Mdc1 and 53BP1 at various time points after irradiation revealed a 70% reduction after 16 h in pachytene and diplotene spermatocytes and round spermatids. Although the DSB repair efficiency is higher then in spermatogonia where only a 40% reduction was found, it still does not compare to somatic cell lines where a 70% reduction occurs in 2 h. Taken together, DNA DSBs repair proteins differ for the various types of spermatogenic cells, no germ cell type possessing the complete set. This likely leads to a compromised efficiency relative to somatic cell lines. From the evolutionary point of view it may be an advantage when germ cells die from DNA damage rather than risk the acquisition of transmittable errors made during the repair process.

Perturbation of the Activity of Replication Origin by Meiosis-specific Transcription

We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in premeiotic S-phase by neutral/neutral two-dimensional gel electrophoresis. The comparison of origin activity of each origin in mitotic and premeiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in premeiotic S-phase. ARS605 is located within the open reading frame of MSH4 gene that is transcribed specifically during an early stage of meiosis. Systematic analysis of relationships between MSH4 transcription and ARS605 origin activity revealed that transcription of MSH4 inhibited the ARS605 origin activity by removing origin recognition complex from ARS605. Deletion of UME6, a transcription factor responsible for repressing MSH4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.

Rad3-Cds1 Mediates Coupling of Initiation of Meiotic Recombination with DNA Replication: Mei4-DEPENDENT TRANSCRIPTION AS A POTENTIAL TARGET OF MEIOTIC CHECKPOINT

Premeiotic S-phase and meiotic recombination are known to be strictly coupled in Saccharomyces cerevisiae. However, the checkpoint pathway regulating this coupling has been largely unknown. In fission yeast, Rad3 is known to play an essential role in coordination of DNA replication and cell division during both mitotic growth and meiosis. Here we have examined whether the Rad3 pathway also regulates the coupling of DNA synthesis and recombination. Inhibition of premeiotic S-phase with hydroxyurea completely abrogates the progression of meiosis, including the formation of DNA double-strand breaks (DSBs). DSB formation is restored in rad3 mutant even in the presence of hydroxyurea, although repair of DSBs does not take place or is significantly delayed, indicating that the subsequent recombination steps may be still inhibited. Examination of the roles of downstream checkpoint kinases reveals that Cds1, but not Chk1 or Mek1, is required for suppression of DSB in the presence of hydroxyurea. Transcriptional induction of some rec+ genes essential for DSB occurs at a normal timing and to a normal level in the absence of DNA synthesis in both the wild-type and cds1delta cells. On the other hand, the transcriptional induction of the mei4+ transcription factor and cdc25+ phosphatase, which is significantly suppressed by hydroxyurea in the wild-type cells, occurs almost to a normal level in cds1delta cells even in the presence of hydroxyurea. These results show that the Rad3-Cds1 checkpoint pathway coordinates initiation of meiotic recombination and meiotic cell divisions with premeiotic DNA synthesis. Because mei4+ is known to be required for DSB formation and cdc25+ is required for activation of meiotic cell divisions, we propose an intriguing possibility that the Rad3-Cds1 meiotic checkpoint pathway may target transcription of these factors.

The Schizosaccharomyces pombe Replication Inhibitor Spd1 Regulates Ribonucleotide Reductase Activity and dNTPs by Binding to the Large Cdc22 Subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.

Meiosis-Specific Regulation of the Saccharomyces cerevisiae S-Phase Cyclin CLB5 Is Dependent on MluI Cell Cycle Box (MCB) Elements in Its Promoter but Is Independent of MCB-Binding Factor Activity

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1.

The Drosophila meiotic kleisin C(2)M functions before the meiotic divisions.

Stepwise and regionally controlled resolution of sister chromatid cohesion is thought to be crucial for faithful chromosome segregation during meiotic divisions. In yeast, the meiosis-specific alpha-kleisin subunit of the cohesin complex, Rec8, is protected from cleavage by separase but only during meiosis I and specifically within the pericentromeric region. While the Drosophila genome does not contain an obvious Rec8 orthologue, as other animal and plant genomes, it includes c(2)M, which encodes a distant alpha-kleisin family member involved in female meiosis. C(2)M associates in vivo with the Smc3 cohesin subunit, as previously shown for yeast Rec8. In contrast to Rec8, however, C(2)M accumulates predominantly after the pre-meiotic S-phase. Moreover, after association with the synaptonemal complex, it disappears again and cannot be detected on meiotic chromosomes by metaphase I. C(2)M cleavage fragments are not observed during completion of the meiotic divisions, and mutations within putative separase cleavage sites do not interfere with meiotic chromosome segregation. Therefore, C(2)M appears to function within the synaptonemal complex during prophase I but possibly not thereafter. This suggests that C(2)M may not confer sister chromatid cohesion needed for meiosis I and II chromosome segregation.

DSC1-MCB regulation of meiotic transcription in Schizosaccharomyces pombe.

Meiosis is initiated from the G1 phase of the mitotic cell cycle, and consists of pre-meiotic S-phase followed by two successive nuclear divisions. Here we show that control of gene expression during pre-meiotic S-phase in the fission yeast Schizosaccharomyces pombe is mediated by a DNA synthesis control-like transcription factor complex (DSC1), which acts upon M lu1 cell cycle box (MCB) promoter motifs. Several genes, including rec8+, rec11+, cdc18+, and cdc22+, which contain MCB motifs in their promoter regions, are found to be co-ordinately regulated during pre-meiotic S-phase. Both synthetic and native MCB motifs are shown to confer meiotic-specific transcription on a heterologous reporter gene. A DSC1-like transcription factor complex that binds to MCB motifs was also identified in meiotic cells. The effect of mutating and over-expressing individual components of DSC1 (cdc10+, res1+, res2+, rep1+ and rep2+) on the transcription of cdc22+, rec8+ and rec11+ during meiosis was examined. We found that cdc10+, res2+, rep1+ and rep2+ are required for correct meiotic transcription, while res1+ is not required for this process. This work demonstrates a role for MCB motifs and a DSC1-like transcription factor complex in controlling transcription during meiosis in fission yeast, and suggests a mechanism for how this specific expression occurs.

Coprinus cinereus DNA ligase I during meiotic development

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA ( CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene ( CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.