Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

Abstract

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.

Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

“…The Smc5ϩ6 complex together with two recently described non-SMC subunits, Nse1 and Nse2, has been shown to mitigate the effects of various forms of DNA damage (Lehmann et al, 1995;Verkade et al, 1999;Fujioka et al, 2002;McDonald et al, 2003;Harvey et al, 2004). Consistently, we found that nse3-1 cells are sensitive to gamma irradiation, UV light, the topoisomerase I poison camptothecin, and replication inhibition by hydroxyurea (Figure 3, A and B).…”

“…It was necessary to overexpress Rad60 from the potent nmt1 promoter to detect the relatively weak interaction with Smc5 and Smc6 (Boddy et al, 2003). It was previously reported that the BRCT domain containing protein, Brc1, is also required for the viability of smc6-X (rad18-X) cells (Verkade et al, 1999). Consistent with codependent functions of Smc5ϩ6 and the Nse proteins, we found that Brc1 is required for the viability of all nse mutants.…”

“…Smc6 of the Smc5ϩ6 heterodimer was initially identified by analysis of radiation-sensitive mutants in fission yeast and Smc5 was subsequently identified by its homology to Smc6 (Lehmann et al, 1995;Verkade et al, 1999;Fousteri and Lehmann, 2000). Like cohesin and condensin, the Smc5ϩ6 complex is essential for viability and seems to control chromatin structure (Lehmann et al, 1995;Harvey et al, 2004).…”

“…Like cohesin and condensin, the Smc5ϩ6 complex is essential for viability and seems to control chromatin structure (Lehmann et al, 1995;Harvey et al, 2004). Studies on mutants of the complex in fission yeast have demonstrated that it has a role in the HR-based repair of DNA damage and that it may also influence checkpoint maintenance (Lehmann et al, 1995;Verkade et al, 1999;McDonald et al, 2003;Harvey et al, 2004). SMC heterodimers need to associate with specific non-SMC family subunits to be functional.…”

“…However, cohesin is recruited to sites of laser-induced DSBs, supporting a more direct role in the repair process (Kim et al, 2002a). The role of condensin in DNA repair is unknown, but a direct involvement is suggested by the finding that a subunit of the complex interacts with DNA ligase IV (Przewloka et al, 2003).Smc6 of the Smc5ϩ6 heterodimer was initially identified by analysis of radiation-sensitive mutants in fission yeast and Smc5 was subsequently identified by its homology to Smc6 (Lehmann et al, 1995;Verkade et al, 1999;Fousteri and Lehmann, 2000). Like cohesin and condensin, the Smc5ϩ6 complex is essential for viability and seems to control chromatin structure (Lehmann et al, 1995;Harvey et al, 2004).…”