The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication. The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway. We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress. mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants. Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage.

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromereassociated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.

TOPBP1interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4 TOPBP1 coprecipitates with Rad3 ATR , suggesting that phosphorylation coordinates formation of an active checkpoint complex. In multicellular eukaryotes, DDRs also interface with the apoptotic and senescence pathways to ensure that specific cell types that receive high levels of damage are removed from the cycling population (Wahl and Carr 2001). Failure of DDRs underlies many cancer-prone human genetic diseases, and mutations in DDR proteins are common events in the etiology of sporadic cancers.Many of the DDRs require the activation of the ATRand ATM-dependent DNA structure checkpoint pathways. Activation of the ATR and ATM kinases promotes a cascade of phosphorylation events. Among these are phosphorylation and activation of two downstream kinases Chk1 and Chk2 (Shiloh 2003). The phosphorylation of target proteins by ATM, ATR, Chk1, and Chk2 results in the regulation of transcription, changes in the profiles of protein stability, and changes to the subcellular localization of certain proteins Shiloh 2003;Yao et al. 2003). Several target proteins have been identified, and in some instances, individual phosphorylation events have been ascribed specific functions. For example, phosphorylation of p53 and its E3 ubiquitin ligase Mdm2 influences the stability and function of p53, an important effector of checkpoint signals (Chene 2003). Because checkpoint pathways are vital for the maintenance of genomic stability and the suppression of carcinogenesis, many studies have aimed to understand the molecular mechanisms underpinning checkpoint pathway activation. Two distinct DNA-structure-responsive checkpoint pathways have been characterized.The ATM-dependent checkpoint responds directly to DNA double-strand breaks (DSBs). Activation of ATM requires the presence of the Mre11-Rad50-Xrs2 NBS1 complex (MRX), and studies in budding yeast demonstrate that the MRX complex binds to DNA doublestrand ends at the site of damage and recruits the ATM homolog Tel1 (Nakada et al. 2003). In human cells, Nbs1 is required for ATM-dependent phosphorylation events in response to DNA damage (Girard et al. 2002;Uziel et al. 2003). ATM phosphorylates MRX in response to DNA damage and also targets a histone H2A variant, several checkpoint mediator proteins (including BRCA1, TOPBP1, P53BP1, and MDC1) and activates Chk2 (Shiloh 2003).The ATR-dependent checkpoint responds to a variety of genotoxic insults, including UV-induced dimers and agents that stall DNA replication. ATR forms a stable protein complex with ATRIP, and this subunit is required to bind ATR-ATRIP to single-stranded DNA

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono-and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.