DNA double-strand breaks are critical lesions that can lead to chromosomal aberrations and genomic instability. In response to DNA damage, Chk1, a serine/threonine kinase, is responsible for cell cycle arrest to prevent damaged cells from progressing through the cell cycle. Here, we report that the disruption of wat1, a WD repeat-containing protein, leads to the phosphorylation of Chk1. The double-deletion of chk1 and wat1 had a grave effect on the survival of fission yeast cells, and the spontaneous recombination rate was also high upon double-deletion of wat1 and chk1, as compared to the single-mutant. In the absence of wat1, the cells exhibited a high level of nuclear fragmentation that resulted in the accumulation of Rad22 yellow fluorescent protein foci. Furthermore, we show that wat1 is required for the regulation of the oxidative stress response. We observed elevated levels of reactive oxygen species (ROS) generation in wat1-null mutant that led to a high degree of propidium iodide staining at nonpermissive temperature. Based on the results presented here, we hypothesize that ROS production in wat1-null mutant cells generates DNA fragmentation that could trigger a checkpoint response and that, in the absence of checkpoint kinase Chk1, the cells exhibit severe growth defects leading to a synthetic lethal phenotype.

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Smc5/6, like cohesin and condensin, is a structural maintenance of chromosomes complex crucial for genome stability. Unlike cohesin and condensin, Smc5/6 carries an essential Nse2 subunit with SUMO E3 ligase activity.

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect and heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

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