Mms22 preserves genomic integrity during DNA replication in Schizosaccharomyces pombe

Abstract

The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important but poorly understood role in preservation of genome integrity. Here we describe a novel gene in Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22 functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability. There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to maintain genomic integrity in a pathway involving Mus81-Eme1.

Figure 1.—

Identification of a putative homolog of MMS22 in S. pombe. (A) A schematic of putative MMS22 homologs in representative fungi showing areas of homology in the N and the C terminus and the corresponding amino acid alignments. The N-terminal homology domain spans S. cerevisiae Mms22p residues 196–292, Coccidioides immitis CIMG_05124 residues 403–499, Aspergillus nidulans AN6261.2 residues 522–614, and S. pombe SPAC6B12.02c (hereafter named Mms22) residues 246–337. The C-terminal homology domain incorporates S. cerevisiae Mms22p residues 1055–1234, CIMG_05124 residues 1615–1815, AN6261.2 residues 1748–1949, and S. pombe Mms22 residues 1319–1517. White text on a solid background indicates amino acid identity and solid text on a shaded background indicates conservative amino acid substitutions. (B) Survival curves of mms22Δ mutants exposed to increasing doses of IR or UV. A total of 500–1000 cells were plated on YES agar in triplicate and immediately exposed to the indicated dose of IR or UV irradiation. Colony numbers were counted following incubation at 30° for 2–3 days and the mean colony number for each dose represented graphically (with untreated normalized to 100% survival). The sensitivity of a rad32Δ mutant was analyzed as positive control. (C) Phenotypes of mms22Δ mutants. Fivefold serial dilutions of cells were plated on YES agar exposed to the indicated DNA-damaging agent and incubated at 30° for 2–3 days.

Figure 2.—

Deletion of Chk1 reduces the elongated phenotype of mms22 mutants. (A) Cells were cultured to midlog phase in YES medium, fixed in 70% ethanol for 10 min at room temperature, and DAPI stained to visualize the nuclei and morphology of the cell. At least 250 cells were scored in three independent experiments and assigned to a particular cell cycle phase as previously described (Noguchi et al. 2004). Mean values were plotted with error bars representing the standard deviation about the mean. (B) Mms22 is not required for checkpoint activation in response to HU. Cells were cultured to midlog phase in YES medium and the culture was split into two. One culture was pelleted, immediately fixed in 70% ethanol for 10 min at room temperature, and DAPI stained, while the other culture was incubated in the presence of 12 mm HU for 4 hr prior to fixation and DAPI staining. Representative images are displayed.

Figure 3.—

Mms22 has checkpoint-independent functions. (A) Phenotypes of mms22 mutants in checkpoint kinase-deficient backgrounds. Fivefold serial dilutions of cells were plated on YES agar exposed to the indicated DNA-damaging agent and incubated at 30° for 2–3 days. (B) Phenotypes of mms22 mutants in checkpoint kinase-deficient backgrounds following acute exposure to UV and HU. For UV exposure, 1000 cells were plated onto triplicate YES agar plates and immediately irradiated with the indicated dose. For survival of acute exposure to HU, midlog phase cells were cultured in YES media containing 12 mm HU for 10 hr. At 0 hr, 1000 cells were plated onto YES agar plates in triplicate and, at the indicated time points, the same culture volume was taken, HU was washed out, and the cells were plated in triplicate. Survival was estimated relative to untreated cells. For all survival assays, recovery was for 2–3 days at 30° unless otherwise stated.

Figure 4.—

Spontaneous DNA damage occurs in the absence of Mms22. (A) Elevated Rad22-YFP foci arise in the S- and G₂-phases of the cell cycle in an mms22Δ background. Cells were cultured to midlog phase in YES medium and imaged live. The numbers of foci in at least 250 nuclei representing different phases of the cell cycle were scored in three independent experiments, and mean values were plotted with error bars representing the standard deviation of the mean. (B) Elevated spontaneous minichromosome 16 loss associated with disruption of Mms22 function. Cells containing the minichromosome are ade⁺ due to allelic complementation. Cells from individual Ade⁺ colonies were plated on adenine-limiting plates and the percentage of chromosome loss events per division was determined. The actual numbers are displayed under the chart (at least half red/total colonies).

Figure 5.—

Mms22 forms nuclear foci that increase in response to DNA damage. (A) Cells expressing ectopic GFP-Mms22 were cultured for 20 hr to midlog phase in selective medium containing thiamine. The culture was split into three, one of which was analyzed immediately as the starting culture, and the other two of which were treated with CPT or DMSO as stated in the materials and methods section. For each culture, foci were scored in at least 250 live nuclei in three independent experiments. A representative data set from one experiment is shown due to variable GFP-Mms22 expression between individual experiments. (B) Mms22 foci represent sites of DSBs following CPT treatment. The majority of spontaneous GFP-Mms22 and Rad22-RFP foci do not colocalize, whereas an increased overlap in signal is observed following DNA damage. Representative images are shown. (C) Quantification of the percentage of GFP-Mms22 foci with an overlapping Rad22-RFP focus before and after CPT treatment. For each culture, foci were scored in three independent experiments and mean values were plotted with error bars representing the standard deviation of the mean.

Figure 6.—

Mms22 functions in a non-HR DNA repair pathway (A) Tetrad dissection of genetic crosses of mms22Δ and HR mutants. Representative spores from three asci are shown for each cross. (B) Synthetic additivity of mms22 and rhp57 mutations. Fivefold serial dilutions of cells were exposed to the indicated DNA-damaging agent and incubated at 30° for 2–3 days.

Figure 7.—

Genetic relationship between Mms22 and other DNA repair proteins. Fivefold serial dilutions of cells were exposed to the indicated DNA-damaging agent and incubated at 30° for 2–3 days.

Figure 8.—

Genetic interactions between Mms22 and components of the replication fork. Fivefold serial dilutions of cells were exposed to the indicated DNA-damaging agent. Plates were incubated at the indicated temperatures for 2–4 days. All DNA-damaging treatments were conducted at 25°.

Figure 9.—

Genetic interactions between Mms22 and DNA polymerases δ and ɛ. Fivefold serial dilutions of cells were exposed to the indicated DNA damaging agent. Plates were incubated at the indicated temperatures for 2–4 days. All DNA-damaging treatments were conducted at 25°.