Fission yeast Hsk1 (Cdc7) kinase is required after replication initiation for induced mutagenesis and proper response to DNA alkylation damage

Abstract

Genome stability in fission yeast requires the conserved S-phase kinase Hsk1 (Cdc7) and its partner Dfp1 (Dbf4). In addition to their established function in the initiation of DNA replication, we show that these proteins are important in maintaining genome integrity later in S phase and G2. hsk1 cells suffer increased rates of mitotic recombination and require recombination proteins for survival. Both hsk1 and dfp1 mutants are acutely sensitive to alkylation damage yet defective in induced mutagenesis. Hsk1 and Dfp1 are associated with the chromatin even after S phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS-sensitive mutants identified a novel truncation allele, rad35 (dfp1-(1-519)), as well as alleles of other damage-associated genes. Although Hsk1-Dfp1 functions with the Swi1-Swi3 fork protection complex, it also acts independently of the FPC to promote DNA repair. We conclude that Hsk1-Dfp1 kinase functions post-initiation to maintain replication fork stability, an activity potentially mediated by the C terminus of Dfp1.

Figure 1.—

Synthetic interactions of hsk1–1312 with replication checkpoint and fork protection complex mutations. (A) hsk1 temperature sensitivity is suppressed by deletion of Δcds1 or Δmrc1 but not Δswi1. (B) MMS sensitivity of hsk1 and dfp1, Δswi1, and Δswi3 single and double mutants. Cells were diluted fivefold on YES under the indicated conditions.

Figure 2.—

Analysis of recombination in hsk1–1312. (A) rad22YFP (FY2878), hsk1–1312 rad22YFP (FY3285), Δswi1 rad22YFP (FY3287), and Δcds1 rad22YFP (FY3286) cultures were grown overnight at 25°. Cultures were split and either no hydroxyurea (async) or 12 mm HU (HU) was added. After 3 hr of growth, cells were washed twice in PBS, and spotted on slides with poly-l-lysine. Images were collected, deconvolved, and counted. Scale bar, 15 μm. (B) Quantification of cell counts averaged from two experiments. (C) Scheme of ade6–his3⁺–ade6 cassette used in mitotic recombination assay. (D) Quantification of mitotic recombination frequency per generation relative to wild type. Wild-type (FY2132), hsk1-1312 (FY3101), swi1(FY3098), and swi3 (FY3100) strains were grown in YES, and two plates of 10⁴ cells were plated to EMM Ade⁻. Survivors were patched to Ade⁻ and Ade⁻ His⁻ to determine histidine prototrophy. Data are the averages of seven or eight independent cultures. Statistical analysis is presented in the text.

Figure 3.—

Hsk1 and Dfp1 bind chromatin during S and G2 phases. (A) Cultures of cdc10 hsk1HA (FY1000), cdc22 hsk1HA (FY1011), and cdc25 hsk1HA (FY1006) were grown at 25°, shifted to 36° for 4 hr, and processed for in situ chromatin binding. Scale bar, 10.7 μm. (B) Quantification of data from A; averages of three experiments are presented and error bars show standard deviations. (C) Cultures of cdc22 dfp1v5 (FY3281) and cdc25 dfp1v5 (FY3282) were grown at 25°, shifted to 36° for 4 hr, and processed for in situ chromatin binding. Scale bar, 10.7 μm. (D) Quantification of data from (C); averages of two experiments are presented and error bars are standard deviations. (E) Hsk1–Dfp1 and Swi1–Swi3 bind chromatin independently. hsk1HA (FY1077), Δswi1 hsk1HA (FY3249), and Δswi3 hsk1HA (FY3251) cultures were grown at 32° and processed for in situ chromatin binding. Scale bar, 10.7 μm. (F) Quantification of data from E; averages of three experiments are presented and error bars show standard deviations.

Figure 4.—

Dfp1ΔC chromatin association with damaged DNA is disrupted. dfp1HA (FY1763) (A) or dfp1ΔCHA (FY1794) (B) cultures were grown at 32°, arrested with 20 mm HU for 3 hr (HU), and released to plain medium (release) or medium with 0.03% MMS (release +MMS) for 1 hr. Cells were then processed for in situ chromatin binding. Scale bar, 10.7 μm. (C) Quantification of data from A and B; averages of two experiments are presented. Error bars show standard deviations.

Figure 5.—

Hsk1–Dfp1 function in the error-prone postreplication repair pathway. (A) hsk1 is required for induced mutagenesis. The graph shows the relative mean forward mutation frequency of the ura4⁺ gene for untreated samples (−) and samples exposed to 0.0025% MMS for 1 hr (+) in wild-type (FY8), Δswi1∷kanMX (FY4588), hsk1–1312 (FY1418), and dfp1^rad35-271 (FY3998) strains. The relative forward mutation frequency is the rate compared to that of wild-type untreated cells. The middle line in each box represents the mean, and the upper and lower limits of the box represent 95% confidence interval calculated from the one-sample t-test. Each confidence interval was calculated from a sample size of at least 12 independently chosen colonies. (B) Synthetic interactions between hsk1 and components of the postreplication repair pathway. Cells were plated in 5× dilutions on YES with the indicated amount of MMS. (C) Synthetic interactions of hsk1–1312, Δswi1, and Δswi3 with Δrhp18 in response to UV. Cultures were grown overnight at 25° into log phase. Cells were plated and plates allowed todry. Plates were exposed to 0, 10, or 25 J/m² UV light. Plates were incubated at 25° for 3–5 days and counted. Values are the average relative viability of three assays; error bars show standard deviations. (D) Synthetic interactions between dfp1–(1-519) and components of the postreplication repair pathway. (E) Synthetic interactions between hsk1 and deletion of the error-prone polymerases.

Figure 6.—

Model for Hsk1 interaction with TLS pathway. The sliding clamp proteins PCNA (green) and 9-1-1 (pink) mediate the choice between repair mechanisms. Homologous recombination is inhibited by the Srs2 helicase and PCNA sumoylation. Polyubiquitination of PCNA by Rhp6/18 and then Mms2/Ubc13/Rad8 drives error free bypass repair. Error prone translesion synthesis by minor polymerases occurs in response to PCNA monoubiquitination. Hsk1 and Dfp1 appear to operate in this pathway independent of Swi1/3 and Rhp6/18. Modified from Kai et al. (2007); Andersen et al. (2008); Branzei et al. (2008).