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.

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The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G 1 . We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G 1 results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G 2 leads to cell death in subsequent metaphase-anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G 1 . Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase.[Key Words: Cell cycle; cohesin; fission yeast; mitosis; UV irradiation; hydroxyurea]Received July 27, 1998; revised version accepted September 9, 1998.In eukaryotic cells, replication of chromosomal DNA occurs once during S phase followed by separation of duplicated DNAs into two daughter nuclei during M-phase. DNA replication is initiated at the onset of S phase in particular chromosomal regions, the origins, which are unwound and stabilized by single-strand-binding protein. The replication machinery is large and complex, containing a number of enzyme subunits including DNA polymerases and helicase. However, other factors are necessary for initiating replication such as the origin recognition complex, which is bound to origin sequences throughout the cell cycle. Cdc6 and the MCM complex are also needed and are loaded onto the origins to form the prereplicative complex (e.g., see Rowles and Blow 1997). Moreover, an essential protein kinase Cdc7/Dbf4 must act prior to replication, so that the replication machinery can interact with the prereplicative complex and thereby ensure continuing replication. The proteins described above are still part of the gene products required for the quality control of DNA replication. When chromosomal DNAs are damaged or replication is blocked, entry into mitosis is restrained until the damage is recognized and repaired (e.g., see Kitazono and Matsumoto 1998). A block of mitotic entry upon damage or inhibition of replication can be accomplished by inactivation of cyclin-dependent protein kinases (CDKs). This checkpoint system is important for maintaining the genome with high fidelity, otherwise damaged chromosomal information will be transmitted to the nuclei of progeny cells.To maintain the gen...

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1 ؉ gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G 1 -S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.Virtually all eukaryotic cells propagate through a process called the cell cycle that consists of four distinct phases, G 1 , S, G 2 , and M, whose principal role is to carry out duplication of the chromosomes and subsequent faithful distribution into daughter cells. Commitment to the initiation of the cell cycle is made at a point in late G 1 phase called start or restriction point. In the fission yeast, Schizosaccharomyces pombe, passage through start requires the execution of at least two regulatory systems, Res-Cdc10-Rep (Res1p-Cdc10p and Res2p-Cdc10p-Rep2p) transcriptional factor complexes and Cdc2p-Cig2p/Cyc17p cyclin-dependent kinase complex (reviewed in references 37 and 54). Res-Cdc10-Rep complexes activate cell cycle start-specific transcription genes, which contain a cis regulatory element called the MluI cell cycle box. One of those target genes is cdc18 ϩ , whose product is a key component of the preinitiation form of origin replication complex and plays a crucial role in loading origins with the replication machinery including DNA polymerases in cooperation with minichromosome maintenance proteins (reviewed in reference 35). Cdc2p-Cig2p activity is also required for origin firing, but its critical target(s) has not been identified yet. These initiation factors and replication factors are highly conserved throughout eukaryotes.In order to ensure faithful transmission of the duplicated c...