Critical functions of Rpa3/Ssb3 in S-phase DNA damage responses in fission yeast

Abstract

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)-binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11Rpa1 and Rad22Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.

Figure 1. Genotoxin sensitive phenotypes of ssb3Δ mutants.

(A) ssb3Δ cells are sensitive to a number of DNA-damaging agents, particularly those that disrupt DNA replication. Tenfold serial dilutions of cells were plated on YES agar medium, exposed to the indicated DNA-damaging agents and incubated at 30°C for 3–4 days. (B) Elimination of Ssb3 causes a weak IR-sensitive phenotype. Mean values of three different experiments are shown, with error bars representing the standard deviation of the mean.

Figure 2. Ssb3 is not required for meiosis.

Wild type x wild type or ssb3Δ×ssb3Δ matings were analyzed by tetrad dissection (left panel). Four-spore asci appeared normal in both matings (right panel).

Figure 3. DNA damage checkpoint responses in ssb3Δ cells.

(A) ssb3Δ cells are elongated even in the absence of DNA damaging agents. Elimination of Chk1 suppresses this elongated phenotype. The mean relative cell lengths of mutants (normalized to wild type = 1.00±0.03 SD) are: ssb3Δ = 1.21±0.04; ssb3Δ chk1Δ = 1.01±0.01; ssb3Δ cds1Δ = 1.21±0.04. Data are derived from 3 independent measurements of 100 cells each (B) ssb3Δ cells arrest division and elongate in response to CPT, showing that the DNA damage checkpoint is intact. The rad3Δ strain is a checkpoint defective control. (C) Chk1 undergoes activating phosphorylation in ssb3Δ cells. After CPT or IR treatment, Chk1 is phosphorylated in ssb3Δ cells as well as in control cells, as indicated by the appearance of a slow-mobility species. Samples were processed for immunoblot analysis of HA-tagged Chk1. (D) Phenotypes of ssb3Δ cells in combination with checkpoint kinase disruptions. Tenfold serial dilutions of cells were plated on YES agar medium, exposed to the indicated DNA-damaging agents, and incubated at 30°C for 3–4 days.

Figure 4. Increased Rad22 DNA repair foci in ssb3Δ cells.

Rad22-YFP foci formation was significantly increased in ssb3Δ cells, especially during the S- and G2-phases of the cell cycle. Control and ssb3Δ cells were cultured in EMM liquid medium at 30°C until mid-log phase, photographed, and the number of nuclei with at least one Rad22-YFP foci was scored. Mean values of three different experiments are shown, with error bars representing the standard deviation of the mean.

Figure 5. Ssb3 localizes to sites of DSBs.

(A) Ssb3 forms nuclear foci that increase in number following DNA damage. Cells expressing endogenous Ssb3-GFP were cultured in YES liquid medium at 30°C until mid-log phase and then treated with CPT or left untreated. Nuclei with at least one or with two or more Ssb3-GFP foci were scored in three independent experiments and the mean values are represented. Error bars correspond to standard deviations of the mean. (B) Ssb3 colocalizes with Rad22. The majority of both spontaneous and CPT-caused Ssb3-GFP foci colocalize with Rad22-RFP foci. Cells expressing endogenous Ssb3-GFP and Rad22-RFP were cultured and CPT-treated as indicated above. Representative images are shown. (C) Quantification of the percentages of nuclei with Ssb3-GFP or Rad22-RFP, with or without CPT treatment. (D) Quantification of the percentages of Ssb3-GFP foci with overlapping Rad22-RFP foci, with or without CPT treatment. In each case, nuclei with foci were scored in three independent experiments and the mean values are plotted, with error bars corresponding to the standard deviation of the mean.

Figure 6. Rad11^Rpa1 foci are increased in ssb3Δ cells.

(A) ssb3Δ rad11-GFP show strong genetic interactions at 30°C. The left panel shows tetrad analysis of a ssb3Δ×rad11-GFP mating germinated at 30°C. The right panel shows a photomicrograph of ssb3Δ rad11-GFP cells from the tetrad dissection plate. (B) Rad11-GFP forms nuclear foci that increase in number following DNA damage even in the absence of Ssb3. Cells expressing endogenous Rad11-GFP in a wild type or ssb3Δ background were cultured in YES liquid medium at 25°C until mid-log phase and then treated with CPT or left untreated. Percentages of nuclei with at least one Rad11-GFP focus are shown. Quantification of Rad11-GFP foci in different cell cycle stages was determined. Foci were scored in three independent experiments and the mean values are represented. Error bars correspond to standard deviations of the mean. (C) Co-localization of Rad11-GFP and Ssb3-RFP foci upon treatment with CPT.

Figure 7. Genetic interactions involving Ssb3 and components of replication fork protection complexes, Okazaki fragment processing, and NER.

Tenfold serial dilutions of cells were exposed to the indicated DNA-damaging agents and plates were incubated at 30°C for 3–4 days, except for rfc3-1 strains which were incubated at 25°C. Representative images of repeat experiments are shown.

Figure 8. Genetic interactions between Ssb3 and Rhp51, Rhp55, Mms22, and Brc1.

Tenfold serial dilutions of cells were exposed to the indicated DNA-damaging agents and plates were incubated at 30°C for 3–4 days. Representative images of repeat experiments are shown.

Figure 9. Genetic interactions between Ssb3 and Mms1, Mus81, Swi5, and Sfr1.

Tenfold serial dilutions of cells were exposed to the indicated DNA-damaging agents and plates were incubated at 30°C for 3–4 days. Representative images of repeat experiments are shown.