Meiotic S-phase damage activates recombination without checkpoint arrest

Abstract

Checkpoints operate during meiosis to ensure the completion of DNA synthesis and programmed recombination before the initiation of meiotic divisions. Studies in the fission yeast Schizosaccharomyces pombe suggest that the meiotic response to DNA damage due to a failed replication checkpoint response differs substantially from the vegetative response, and may be influenced by the presence of homologous chromosomes. The checkpoint responses to DNA damage during fission yeast meiosis are not well characterized. Here we report that DNA damage induced during meiotic S-phase does not activate checkpoint arrest. We also find that in wild-type cells, markers for DNA breaks can persist at least to the first meiotic division. We also observe increased spontaneous S-phase damage in checkpoint mutants, which is repaired by recombination without activating checkpoint arrest. Our results suggest that fission yeast meiosis is exceptionally tolerant of DNA damage, and that some forms of spontaneous S-phase damage can be repaired by recombination without activating checkpoint arrest.

Figure 1.

Checkpoint proteins do not arrest meiotic divisions in response to exogenous DNA damage. (A) Diploid cultures were released into synchronous meiosis in the presence or absence of camptothecin (CPT) or methylmethane sulfonate (MMS) and analyzed for meiotic progression. The proportion of cells having undergone no meiotic divisions (1 nucleus, circles), MI (2 nuclei, squares) or MII (3+ nuclei, triangles) were scored at various timepoints by DAPI microscopy. 300 cells were counted per timepoint. Divisions in Δcds1 cells are slightly accelerated relative to wild-type in the absence of DNA damage; Δrad3 cells behave similarly to Δcds1 cells (data not shown). (B) 300 cells from the ten hour timepoints shown in (A) were scored for normal and abnormal asci formation by DAPI microscopy and results represented as the percent average of total cells. (C) Cds1 and Chk1 protein phosphorylation in response to meiotic DNA damage was analyzed by anti-HA western blot. Diploids expressing Cds1-HA or Chk1-HA were sampled for protein during vegetative growth (asyn.), overnight G1 arrest (0 h timepoint), and 4 h after the induction of synchronous meiosis in the presence or absence of 5mU/ml bleomycin (bleo.), 0.005% MMS, or 15 mM hydroxyurea (HU). Chk1 protein from vegetative Δcds1 diploids in HU for 3 h and in meiosis for 5 h is included as a phosphorylation control, as well as Chk1 protein from vegetative wild-type cells treated with 0.1% MMS for one hour. CPT treated cells complete meiS by 3 h while HU treated cells remain arrested with 2C DNA content beyond 5 h by FACS (data not shown). PCNA levels are included as a loading control.

Figure 4.

Checkpoint mutants sustain increased meiotic recombination without loss of spore viability. (A) Reduced spore viability in a Δrec12 background is partially rescued by loss of checkpoint proteins. Checkpoint deficient Δrec12 parental strains were mated and microdissected asci assayed for colony formation on rich medium. Average viability (y-axis) in 80 asci from four independent cross-es was analyzed for each double mutant and Δrec12 single mutant progeny. Error bars =± 1 SD. Viability is significantly elevated in Δcds1 Δrec12 (p-value = 0.021), Δchk1 Δrec12 (p-value = 0.008), and Δrad3 Δrec12 (p-value = 0.005) (asterisks) relative to Δrec12, whereas Δtel1 Δrec12 viability is not (p-value = 0.529). (B) Percent of ade+ recombinant progeny (y-axis) recovered in cross-es of ade-checkpoint mutants; ade prototrophy is indicative of sister chromatid exchange (SCE). A minimum of 5600 viable spores from four independent cross-es of each checkpoint mutant and wild-type (wt) were assayed for ade prototrophy; error bars = ± 1 SD. SCE is statistically elevated in Δtel1 (p-value = 0.033) and Δcds1 (p-value = 0.017) (asterisks) but not Δchk1 (p-value = 0.146) or Δrad3 (p-value = 0.185) mutants relative to wild-type levels. (C) Spore viability as assayed by tetrad dissection onto rich media (50 asci per mutant) is listed as a percentage of theoretical maximum viability (four spores per ascus).

Figure 2.

Meiotic cells assemble MI spindles in the presence of Rhp51 foci. Indirect immunofluorescence against α-tubulin and Rhp51 was performed on ethanol fixed diploid cells released into synchronous meiosis in the presence or absence of 15 mM hydroxyurea (+HU) or 0.01% methylmethane sulfonate (+MMS). Cells with assembled MI spindles (tubulin, red) were examined for the presence of Rhp51 foci (green) on DNA (DAPI, blue). Representative MI cells with (wild-type +MMS, Δcds1 +HU) and without prominent Rhp51 foci (wild-type) are shown. A total of 100 cells with assembled MI spindles per condition from two independent blinded experiments were counted; percentages represent the average number of MI cells with prominent Rhp51 foci from both experiments. 34% of untreated Δcds1 cells and 39% of untreated Δchk1 cells with MI spindles showed Rhp51 foci. Scale bar = 10 μm.

Figure 3.

Replication checkpoint mutants sustain increased DNA damage during meiotic S-phase. (A) Immunofluorescence against phosphorylated histone H2A (γH2AX, green) was performed on chromosome spreads prepared hourly from diploid cultures released into synchronous meiosis in the presence or absence of 15 mM hydroxyurea (HU). DAPI staining for DNA is shown in blue; phospho-H2A localizes to DNA (merge). Representative spreads with less (wild-type, 2 h) or more (Δcds1, 2 h) than 50% of visible DNA immunoreactive to anti-γH2AX are shown. Scale bar = 10 μm. (B) 100 spreads per timepoint from three independent timecourses were quantitated by blinded counts for phospho-H2A immunoreactivity. The average number of spreads with greater than 50% (stippled bars) or <50% (white bars, stacked; height of both bars represents total percentage of immunoreactive spreads) of DNA area showing γH2AX immunoreactivity is indicated at each timepoint; error bars = ± 1 SD. Approximately 20% of Δcds1 cells in an unperturbed meiotic S-phase show γH2AX immunoreactivity analogous to what is observed in the majority of Δcds1 cells under replication block. The majority of cells in all three time-courses were in S-phase at 2 h and had completed meiS by 3 h, as determined by FACS analysis (data not shown).