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) protein-docking 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.

FIG. 1

Identification of Mus81. (A) Cds1 is important for DNA damage tolerance. The uve1 rad13 and cds1 uve1 rad13 strains were assayed for UV survival (all results shown for UV sensitivity assays are representative examples of two or more experiments). (B) The cds1-fha* allele impairs DNA damage tolerance. uve1 rad13 and cds1-fha* uve1 rad13 cells were assayed for UV survival. (C) Schematic representation of Mus81 from S. pombe (SpMus81) and S. cerevisiae (ScMus81) and human XPF (HuXPF) (572, 632, and 905 amino acids, respectively). Percent identities between putative endonuclease (endo) and helix-hairpin-helix (H) domains are shown. The nonconserved helicase domain of XPF is shown (helic). Light shading depicts regions sharing more than 27% identity. (D) Alignment of endonuclease domains of Mus81 homologs and XPF family members. Sp, S. pombe; Sc, S. cerevisiae; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster, Hu, human. Shading highlights homologous residues, and the boxes show identities (PSI-BLAST results; alignment with ClustalW). (E) Alignment of the helix-hairpin-helix domains of Mus81 homologs and XPF family members.

FIG. 2

Mus81 and Cds1 associate in vivo. (A) Cells that expressed Mus81:myc and Cds1:HA from genomic loci were treated (+) or not treated (−) with HU. Immunoprecipitation with myc antibodies showed that Mus81:myc coprecipitated with Cds1:HA (WT) and Cds1 kinase dead (K.D.) but not with the Cds1 FHA mutant (FHA*). A Cds1 deletion strain (▵) served as a control. The bottom panel (total) is an immunoblot of Cds1:HA present in samples prior to immunoprecipitation. Note that lower-mobility forms of Mus81:myc were detected only in wild-type cells. (B) The 1-to-190 region of the Cds1 wild type (WT) or the FHA mutant (FHA*) was expressed from the nmt1 promoter as a GST fusion protein (GST:Cds1¹⁹⁰) in a Mus81:HA strain. GST:Cds1¹⁹⁰ proteins were purified and detected with amido black or immunoblotted with antibodies to HA. Mus81:HA coprecipitated with the wild-type but not with the mutant FHA domain. (C) Mus81 is a phosphoprotein. A Mus81:13myc strain was treated with HU (+) or not treated. (−) A Mus81:myc strain was immunoprecipitated and treated with λ phosphatase (+) or not treated, (−) either with (+) or without (−) the phosphatase inhibitor vanadate.

FIG. 3

Mus81 is important for tolerance of UV damage. (A) UV survival is impaired in a mus81 mutant. UV survival rates of wild-type, chk1, mus81, cds1 chk1, mus81 chk1, and mus81 cds1 chk1 cells were measured. (B) The mus81 mutation diminishes UV survival in a NER-defective rad13 strain. Wild-type, mus81, rad13, and mus81 rad13 cells were tested for UV survival. (C) The mus81 mutation impairs UV survival in a UVER-defective uve1 strain. Wild-type, mus81, uve1, and mus81 uve1 cells were tested for UV survival. (D) Mus81 contributes to UV survival in the absence of NER and UVER. mus81, uve1 rad13, and mus81 uve1 rad13 cells were assayed for UV survival. (E) Mus81 appears to function in a Cds1-dependent UV tolerance pathway. cds1 uve1 rad13, mus81 uve1 rad13, and cds1 mus81 uve1 rad13 cells were assayed for UV survival. (F) Mus81 appears to function in a Rad3-dependent pathway for UV survival. Wild-type, mus81, rad3, and rad3 mus81 cells were assayed for UV survival. All results shown for UV sensitivity assays are representative of two or more experiments.

FIG. 4

Mus81 is important for survival under conditions that stall replication forks. (A) mus81 cells are sensitive to HU. Serial 10-fold dilutions (10⁴ to 10¹) of cells were incubated on agar medium supplemented with no HU or 5 mM HU. (B) Cds1 is activated normally by HU treatment in mus81 cells. Wild-type (WT), mus81, and cds1 strains were incubated in the presence (+) or absence (−) of HU for 3 h. Cds1 activity was measured with the GST:Wee1¹⁵² substrate as previously described (6). SDS, sodium dodecyl sulfate. (C) The mus81 mutation lowers the restrictive temperature of thermosensitive DNA polymerase delta (polδ^ts) (cdc6-23) and alpha (polα^ts) (pol1-1) alleles, shown at 28 and 33°C, respectively. The mus81 mutation does not lower the restrictive temperature of a polymerase epsilon (polɛ^ts) (cdc20-m10) allele, shown at 33°C.

FIG. 5

(A) Role of Mus81 in UV tolerance involves Rhp51-dependent recombination repair. Wild-type, mus81, rhp51, and rhp51 mus81 cells were tested for UV resistance. (B) Mus81 mutants are not significantly sensitive to ionizing radiation. Wild-type, mus81, and rhp51 cells were tested for resistance to ionizing radiation. All results shown for damage sensitivity assays are representative of two or more experiments. (C) mus81 cells are viable but display Rad3-dependent cell elongation. Mutant mus81 or mus81 rad3 cells were grown at 30°C in YES media, fixed in ethanol, and stained with DAPI to visualize DNA (right panels). Nomarski images are shown in the left panels. (D) Meiosis defect of Mus81. Wild-type diploids or diploids homozygous for mus81 or rhp51 were sporulated and plated on YES media to determine spore viability. Values are given as means ± standard deviations.