Translesion synthesis polymerases contribute to meiotic chromosome segregation and cohesin dynamics in Schizosaccharomycespombe.

Translesion synthesis polymerases (TLSPs) are non-essential error-prone enzymes that ensure cell survival by facilitating DNA replication in the presence of DNA damage. In addition to their role in bypassing lesions, TLSPs have been implicated in meiotic double-strand break repair in several systems. Here, we examine the joint contribution of four TLSPs to meiotic progression in the fission yeast Schizosaccharomyces pombe. We observed a dramatic loss of spore viability in fission yeast lacking all four TLSPs, which is accompanied by disruptions in chromosome segregation during meiosis I and II. Rec8 cohesin dynamics are altered in the absence of the TLSPs. These data suggest that the TLSPs contribute to multiple aspects of meiotic chromosome dynamics.

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology.

Deregulation of mini-chromosome maintenance (MCM) proteins is associated with genomic instability and cancer. MCM complexes are recruited to replication origins for genome duplication. Paradoxically, MCM proteins are in excess than the number of origins and are associated with chromatin regions away from the origins during G1 and S phases. Here, we report an unusually wide left-handed filament structure for an archaeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation. The filament has a remarkably strong electro-positive surface spiraling along the inner filament channel for DNA binding. We show that this MCM filament binding to DNA causes dramatic DNA topology change. This newly identified function of MCM to change DNA topology may imply a wider functional role for MCM in DNA metabolisms beyond helicase function. Finally, using yeast genetics, we show that the inter-subunit interactions, important for MCM filament formation, play a role for cell growth and survival.

Rapid regulation of nuclear proteins by rapamycin-induced translocation in fission yeast.

Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically, this exploits temperature-sensitive mutations or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the anchor-away technique, originally designed in budding yeast by Laemmli lab. This method relies on a rapamycin-mediated interaction between the FRB- and FKBP12-binding domains to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof of principle.

Analysis of transcriptional response in haploid and diploid Schizosaccharomyces pombe under genotoxic stress.

Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions.

Chromodomain mutation in S. pombe Kat5/Mst1 affects centromere dynamics and DNA repair.

KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that is involved in multiple cellular activities. This family is characterized in part by containing a chromodomain, a motif associated with binding methylated histones. We show that a chromodomain mutation in the S. pombe Kat5, mst1-W66R, has defects in pericentromere silencing. mst1-W66R is sensitive to camptothecin (CPT) but only at an increased temperature of 36°C, although it is proficient for growth at this temperature. We also describe a de-silencing effect at the pericentromere by CPT that is independent of RNAi and methylation machinery. We also show that mst1-W66R disrupts recruitment of proteins to repair foci in response to camptothecin-induced DNA damage. Our data suggest a function of Mst1 chromodomain in centromere heterochromatin formation and a separate role in genome-wide damage repair in CPT.

The CINs of the centromere.

Replication stress is a significant contributor to genome instability. Recent studies suggest that the centromere is particularly susceptible to replication stress and prone to rearrangements and genome damage, as well as chromosome loss. This effect is enhanced by loss of heterochromatin. The resulting changes in genetic organization, including chromosome loss, increased mutation and loss of heterozygosity, are important contributors to malignant growth.

Multiple DNA repair pathways contribute to MMS-induced post-replicative DNA synthesis in S. pombe .

Replication stress can induce DNA synthesis outside of replicative S-phase. We have previously demonstrated that fission yeast cells stimulate DNA synthesis in G2-phase but not in M-phase in response to DNA alkylating agent MMS. In this study, we show that various DNA repair pathways, including translesion synthesis and break-induced replication contribute to post-replicative DNA synthesis. Checkpoint kinases, various repair and resection proteins, and multiple polymerases are also involved.

Impact of 1,6-hexanediol on Schizosaccharomyces pombe genome stability.

Phase separation is a major mechanism of macromolecular condensation within cells. A frequently chosen tool for global disruption of phase separation via weak hydrophobic interactions is treatment with 1,6-hexanediol. This study evaluates the cytotoxic and genotoxic effects of treating live fission yeast with 1,6-hexanediol. We find that 1,6-hexanediol causes a drastic decrease in cell survival and growth rate. We also see a reduction in HP1 protein foci and increase in DNA damage foci. However, there is no evidence for increased genomic instability in two classically phase-separated domains, the heterochromatic pericentromere and the nucleolar rDNA repeats. This study reveals that 1,6-hexanediol is a blunt tool for phase separation inhibition and its secondary effects must be taken into consideration during its in vivo use.

Replication stress by MMS stimulates DNA synthesis in post-replicative G2-phase in S. pombe .

DNA replication is generally limited to S-phase but replication stress can drive cells to undergo DNA synthesis outside of S-phase. Mitotic DNA synthesis pathway is known to be activated to deal with replication stress-induced chromosomal instability. There is also growing evidence that residual DNA synthesis can occur in G2. We demonstrate that fission yeast cells stimulate DNA synthesis in G2-phase but not in M-phase in response to DNA alkylating agent MMS. Auxin-induced degradation of DNA replication helicase Mcm4 during G2, but not during mitosis, inhibits post-replicative DNA synthesis.

Revised fission yeast gene and allele nomenclature guidelines for machine readability.

Standardized nomenclature for genes, gene products, and isoforms is crucial to prevent ambiguity and enable clear communication of scientific data, facilitating efficient biocuration and data sharing. Standardized genotype nomenclature, which describes alleles present in a specific strain that differ from those in the wild-type reference strain, is equally essential to maximize research impact and ensure that results linking genotypes to phenotypes are Findable, Accessible, Interoperable, and Reusable (FAIR). In this publication, we extend the fission yeast clade gene nomenclature guidelines to support the curation efforts at PomBase (www.pombase.org), the Schizosaccharomyces pombe Model Organism Database. This update introduces nomenclature guidelines for noncoding RNA genes, following those set forth by the Human Genome Organisation Gene Nomenclature Committee. Additionally, we provide a significant update to the allele and genotype nomenclature guidelines originally published in 1987, to standardize the diverse range of genetic modifications enabled by the fission yeast genetic toolbox. These updated guidelines reflect a community consensus between numerous fission yeast researchers. Adoption of these rules will improve consistency in gene and genotype nomenclature, and facilitate machine-readability and automated entity recognition of fission yeast genes and alleles in publications or datasets. In conclusion, our updated guidelines provide a valuable resource for the fission yeast research community, promoting consistency, clarity, and FAIRness in genetic data sharing and interpretation.

Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase.

The minichromosome maintenance (MCM) complex, a replicative helicase, is a heterohexamer essential for DNA duplication and genome stability. We identified Schizosaccharomyces pombe mcb1(+) (Mcm-binding protein 1), an apparent orthologue of the human MCM-binding protein that associates with a subset of MCM complex proteins. mcb1(+) is an essential gene. Deletion of mcb1(+) caused cell cycle arrest after several generations with a cdc phenotype and disrupted nuclear structure. Mcb1 is an abundant protein, constitutively present across the cell cycle. It is widely distributed in cytoplasm and nucleoplasm and bound to chromatin. Co-immunoprecipitation suggested that Mcb1 interacts robustly with Mcm3-7 but not Mcm2. Overproduction of Mcb1 disrupted the association of Mcm2 with other MCM proteins, resulting in inhibition of DNA replication, DNA damage, and activation of the checkpoint kinase Chk1. Thus, Mcb1 appears to antagonize the function of MCM helicase.

Determinants of RPA megafoci localization to the nuclear periphery in response to replication stress.

Upon replication stress, ssDNA, coated by the ssDNA-binding protein RPA, accumulates and generates a signal to activate the replication stress response. Severe replication stress induced by the loss of minichromosome maintenance helicase subunit Mcm4 in the temperature-sensitive Schizosaccharomyces pombe degron mutant (mcm4-dg) results in the formation of a large RPA focus that is translocated to the nuclear periphery. We show that resection and repair processes and chromatin remodeler Swr1/Ino80 are involved in the large RPA foci formation and its relocalization to nuclear periphery. This concentrated accumulation of RPA increases the recruitment of Cds1 to chromatin and results in an aberrant cell cycle that lacks MBF-mediated G1/S accumulation of Tos4. These findings reveal a distinct replication stress response mediated by localized accumulation of RPA that allows the evasion of cell cycle arrest.

Hsk1-Dfp1 is required for heterochromatin-mediated cohesion at centromeres.

Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.

Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double-strand break.

Chromatin remodeling is essential for effective repair of a DNA double-strand break (DSB). KAT5 (Schizosaccharomyces pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA DSB, including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination (HR). These phenotypes of mst1 are similar to pht1-4KR, a nonacetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs toward HR pathways by modulating resection at the DSB.

Monitoring Schizosaccharomyces pombe genome stress by visualizing end-binding protein Ku.

Studies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

A visual atlas of meiotic protein dynamics in living fission yeast.

Meiosis is a carefully choreographed dynamic process that re-purposes proteins from somatic/vegetative cell division, as well as meiosis-specific factors, to carry out the differentiation and recombination pathway common to sexually reproducing eukaryotes. Studies of individual proteins from a variety of different experimental protocols can make it difficult to compare details between them. Using a consistent protocol in otherwise wild-type fission yeast cells, this report provides an atlas of dynamic protein behaviour of representative proteins at different stages during normal zygotic meiosis in fission yeast. This establishes common landmarks to facilitate comparison of different proteins and shows that initiation of S phase likely occurs prior to nuclear fusion/karyogamy.

Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation.

BACKGROUND: Histone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. RESULTS: We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Deltaelp3 mutant. We examined genetic interactions between Deltaelp3 and two other HAT mutants, Deltamst2 and Deltagcn5 and used whole genome microarray analysis to analyze their effects on gene expression. CONCLUSIONS: Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.

Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant.

Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast.

From yeast to humans, the cell cycle is tightly controlled by regulatory networks that regulate cell proliferation and can be monitored by dynamic visual markers in living cells. We have observed S phase progression by monitoring nuclear accumulation of the FHA-containing DNA binding protein Tos4, which is expressed in the G1/S phase transition. We use Tos4 localization to distinguish three classes of DNA replication mutants: those that arrest with an apparent 1C DNA content and accumulate Tos4 at the restrictive temperature; those that arrest with an apparent 2C DNA content, that do not accumulate Tos4; and those that proceed into mitosis despite a 1C DNA content, again without Tos4 accumulation. Our data indicate that Tos4 localization in these conditions is responsive to checkpoint kinases, with activation of the Cds1 checkpoint kinase promoting Tos4 retention in the nucleus, and activation of the Chk1 damage checkpoint promoting its turnover. Tos4 localization therefore allows us to monitor checkpoint-dependent activation that responds to replication failure in early vs. late S phase.