Damage Tolerance Protein Mus81 Associates with the FHA1 Domain of Checkpoint Kinase Cds1

Abstract

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) proteindocking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.Genome integrity is vulnerable during DNA replication. The act of replication can convert a relatively benign single-strand DNA break to a cytotoxic double-strand break. A cyclobutane dimer, formed by UV irradiation, is sufficient to block the progression of DNA polymerases and to cause replication fork collapse (17). To cope with these problems, eukaryotic organisms have developed mechanisms for replicating DNA through and around damage in ways that cause minimal genome instability. Notable among the proteins involved in these processes are lesion bypass polymerases that can replicate through cyclobutane dimers (45). Recombination and double-strand break repair enzymes are also important, participating in the direct repair of DNA structure abnormalities that arise during DNA replication or permitting continued replication around sites of damage (17). These systems, and perhaps others that remain undiscovered, collectively form a genome defense system that allows tolerance of damage during DNA replication.In the fission yeast Schizosaccharomyces pombe, the checkpoint kinase Cds1 is thought to regulate DNA damage tolerance systems (26,28,31). Cds1 is the presumptive homolog of Rad53 in the budding yeast Saccharomyces cerevisiae and Cds1 (also called Chk2) in humans (7,9,16,27). One of its functions is to delay the onset of mitosis when genome replication encounters difficulties (8,26,47). Cds1 enforces this S-M checkpoint by regulating the Cdc25 and Mik1 mitotic control proteins (2,8,18,47). In addition, Cds1 regulates the way in which DNA is replicated under conditions that cause replicational stress. For example, Cds1 is required to slow the replication of DNA that has been damaged by UV irradiation (26,34). A similar function has been ascribed to Rad53 in budding yeast (32). Slowing of DNA replication partly involves the suppression of late-firing replication origins (35, 37). Activation of C...

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

“…Mus81-Eme1 is required in situations that cause replication fork pausing or collapse (reviewed in Heyer et al, 2003;Kai and Wang, 2003). For example, Mus81 mutation severely lowers the restrictive temperature of thermosensitive alleles of DNA polymerases ␣ and ␦ (Boddy et al, 2000). Furthermore, Mus81 mutants are hypersensitive to chemical agents known to arrest or collapse replication forks such as hydroxyurea, camptothecin (topoisomerase I poison), and methylmethane sulfonate (reviewed in Heyer et al, 2003;Kai and Wang, 2003).…”

“…Several lines of evidence suggest that Mus81-Eme1 processes replication-associated structures that form when replication forks stall. Fission yeast Mus81 was identified by virtue of its specific interaction with the replication checkpoint kinase Cds1 (Boddy et al, 2000). Mus81-Eme1 is required in situations that cause replication fork pausing or collapse (reviewed in Heyer et al, 2003;Kai and Wang, 2003).…”

“…Immunoblotting was performed as described using extracts made from cells lysed in a bead beater (Boddy et al, 2000). Briefly, cells were lysed in buffer A (50 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 5 g/ml each of leupeptin, pepstatin, and aprotinin, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and resolved in 10% SDS-PAGE.…”

“…UV and ionizing radiation sensitivity assays were performed as described previously (Boddy et al, 2000). For hydroxyurea (HU; Sigma-Aldrich, St. Louis, MO) and camptothecin (CPT; Sigma-Aldrich) sensitivity assays, plates were supplemented with the indicated concentrations of drug.…”

Slx1-Slx4 Are Subunits of a Structure-specific Endonuclease That Maintains Ribosomal DNA in Fission Yeast

“…The sgs1 phenotype is characterized by an increase in intra-and interchromosomal recombination, especially at the tandem repeated ribosomal DNA (rDNA) loci (Watt et al, 1995;Sinclair and Guarente, 1997), whereas rqh1 mutants are unable to segregate their chromosomes properly under conditions that stall replication (Stewart et al, 1997). Interestingly, Sgs1 and Rqh1 are essential for viability in the absence of the Mus81-Mms4 and Mus81-Eme1 complexes, respectively (Boddy et al, 2000;Mullen et al, 2001). Mus81 complexes are structure-specific endonucleases that process recombination intermediates formed during meiotic recombination or that arise from fork arrest or collapse Kaliraman et al, 2001).…”