DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle-and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.Genetic inheritance requires exceptional genetic stability over many generations of cells and organisms. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell cycle machinery with a series of surveillance pathways, termed checkpoints, that provide the cells with the capacity to survive genotoxic insults. These protective mechanisms are signal transduction pathways specialized in detecting abnormal DNA structures and in coordinating cell cycle progression with DNA repair. Their activation leads to cell cycle progression delay and concomitant activation of DNA repair pathways, thus preventing replication or segregation of damaged DNA molecules.The keystone of the DNA damage checkpoint is a protein kinase family related to phosphoinositide 3-kinase, among which are Saccharomyces cerevisiae Mec1 (48,61,90) and Tel1 (26, 52), Schizosaccharomyces pombe Rad3 (5), Drosophila melanogaster Mei-41 (29), and mammalian ATR (5) and ATM (72). These protein kinases respond to various stresses by phosphorylating key proteins, thus regulating numerous processes, depending on the spectra of their substrates (for reviews, see references 1 and 75). In particular, S. cerevisiae Mec1 and S. pombe Rad3, more closely related to human ATR, are the prototype transducers of the DNA damage and replication stress signals; they respond to UV damage, double-strand breaks (DSBs), and stalled replication forks. Conversely, yeast Tel1, similar to human ATM, is likely involved only in the response to DSBs (for reviews, see references 57 and 75).Tel1 and Mec1 also contribut...

The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a condition characterized by excessive deposition of extracellular matrix. Fibrogenic cells, including hepatic stellate cells and myofibroblasts, are activated in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin (AR) is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. Here, we have addressed the in vivo role of AR in experimental liver fibrosis. To this end, liver fibrosis was induced in AR؉/؉ and AR؊/؊ mice by chronic CCl 4 administration. Histological and molecular markers of hepatic fibrogenesis were measured. Additionally, the response of cultured human and mouse liver fibrogenic cells to AR was evaluated. We observed that AR was expressed in isolated Kupffer cells and liver fibrogenic cells in response to inflamatory stimuli and platelet-derived growth factor, respectively. We demonstrate that the expression of ␣-smooth muscle actin and collagen deposition were markedly reduced in AR؊/؊ mice compared to AR؉/؉ animals. AR؊/؊ mice also showed reduced expression of tissue inhibitor of metalloproteinases-1 and connective tissue growth factor, two genes that responded to AR treatment in cultured fibrogenic cells. AR also stimulated cell proliferation and exerted a potent antiapoptotic effect on isolated fibrogenic cells. Conclusion: These results indicate that among the different EGFR ligands, AR plays a specific role in liver fibrosis. AR may contribute to the expression of fibrogenic mediators, as well as to the growth and survival of fibrogenic cells. Additionally, our data lend further support to the role of the EGFR system in hepatic fibrogenesis. ( Pio XII, n55, 31008 Pamplona, Spain. E-mail: maavila@unav.es (M.A.A.) and cberasain@unav.es (C.B.); fax: 34-948-194717.

SummaryWe have cloned chs1, a Schizosaccharomyces pombe gene with similarity to class II chitin synthases, and have shown that it is responsible for chitin synthase activity present in cell extracts from this organism. Analysis of this activity reveals that it behaves like chitin synthases from other fungi, although with speci®c biochemical characteristics. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth. In contrast, chs1 expression increases signi®cantly during sporulation, and this is accompanied by an increase in chitin synthase activity. In addition, spore formation is severely affected when both parental strains carry a chs1 deletion, as a result of a defect in the synthesis of the ascospore cell wall. Finally, we show that wild-type, but not chs1 À /chs1 À , ascospore cell walls bind wheatgerm agglutinin. Our results clearly suggest the existence of a relationship between chs1 , chitin synthesis and ascospore maturation in S. pombe.

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DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/ activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.Chromosomal breaks can occur at unpredictable locations in the genome of eukaryotic cells as a result of ionizing radiation, radiomimetic chemicals, or DNA replication across nicked DNA. Moreover, they are introduced in a programmed manner to initiate meiotic recombination during meiosis, the specialized differentiation process in which two rounds of chromosome segregation follow one round of DNA replication (reviewed in references 21 and 26). In the first meiotic division (meiosis I) homologous chromosomes pair and separate, whereas sister chromatids segregate from each other in the second division (meiosis II). Both unprogrammed and programmed double-strand breaks (DSBs) can be repaired by homologous recombination. The primary function of homologous recombination in mitotic cells is to repair DSBs, whereas, during meiosis, it is essential to establish a physical connection between homologous chromosomes, thus ensuring their correct pairing and subsequent segregation at the first meiotic division. In any case, for crossovers to be functional in promoting meiosis I execution in Saccharomyces cerevisiae, they must occur in the context of a proteinaceous tripartite structure, the synaptonemal complex, which connects the axes of homologs along their entire lengths via a close-packed array of transverse filaments (reviewed in reference 36).Programmed meiotic DSB formation requires the product of the meiosis-specific gene SPO11, which, together with several other factors, breaks both strands of a DNA molecule, creating a DSB with covalent linkages between the newly created 5Ј DNA ends and a Spo11 catalytic tyrosine residue (18,19). In S. cerevisiae, the highly conserved Sae2 protein and the MRX (Mre11-Rad50-Xrs2) complex catalyze the endonucleolytic cleava...