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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. [ABSTRACT FROM AUTHOR]

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.

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 temperaturesensitive 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. [ABSTRACT FROM AUTHOR]

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esal and human Tip60 (KAT5). We have isolated a temperaturesensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole, mst1 has an increased frequency of Rad22 repair loci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hipl (Sc Hirl), and the Msc1 protein related to a family of histone demethylases, mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.

In Schizosaccharomyces pombe, three genes, [sir2.sup.+], [hst2.sup.+], and [hst4.sup.+], encode members of the Sir2 family of conserved [NAD.sup.+]-dependent protein deacetylases. The S. pombe [sir2.sup.+] gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, [sir2.sup.+] is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe [sir2.sup.+], suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit [cut9.sup.+], the initiation/checkpoint factor [rad4.sup.+]/[cut5.sup.+], and the first mutant allele of [psf2.sup.+], a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AA[A.sup.+] ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.

MCM proteins are required for the proper regulation of DNA replication. We cloned fission yeast [mcm.sup.7+] and showed it is essential for viability; spores lacking [mcm.sup.7+] begin S phase later than wild-type cells and arrest with an apparent 2C DNA content. We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, [cdc.sup.23+]. mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay. We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele. However, cdc23-M36interacts with mcm4ts. Unlike other mcm mutants or cdc23, mcm7-98is synthetically lethal with checkpoint mutants [DELTA]cds1, [DELTA]chk1, or [DELTA]rad3, suggesting chromosomal defects even at permissive temperature. Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins. Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.

A mutation within the Schizosaccharomyces pombe [cdc24.sup.+] gene was identified previously in a screen for cell division cycle mutants and the [cdc24.sup.+] gene was determined to be essential for S phase in this yeast. We have isolated the [cdc24.sup.+] gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking [cdc24.sup.+] are inviable, indicating that [cdc24.sup.+] is an essential gene. The transcript of [cdc24.sup.+] is present at constant levels throughout the cell cycle. Cells lacking [cdc24.sup.+] function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

The [cdc19.sup.+] gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between [cdc19.sup.+] and genes encoding subunits of DNA polymerase [Delta] and the replication initiator [cdc18.sup.+]. We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

The gene functions, cdc2+ and cdc10+, are required during the commitment control point START, a phase responsible for delaying the onset of mitosis until the completion of S phase of the cell cycle. The major role played by gene cdc18+ in chaining the S phase to START and mitosis is clearly demonstrated. The cdc2+ gene encodes the p34 (cdc2) protein kinase which plays a functional role in initiating mitosis. The START gene cdc10+ encodes an 85kd protein, p85 (cdc10) comprising a part of the transcription factor complex responsible for the activation of specific genes during the starting stages of the S phase.