doi: 10.1534/genetics.112.145730. Epub 2012 Nov 12.
A mammalian-like DNA damage response of fission yeast to nucleoside analogs
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- PMID:23150603
- PMCID: PMC3527242
- DOI: 10.1534/genetics.112.145730
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A mammalian-like DNA damage response of fission yeast to nucleoside analogs
Sarah A Sabatinos et al. Genetics.2013 Jan.
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Abstract
Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.
Figures
BrdU and EdU doses affect signal, viability, and cell division. (A) Time course of incorporation at 32.6 or 326 µM BrdU in HU-synchronized cells after release. Asynchronous (AS) cultures were blocked for 4 hr in HU (HU time point) before release at 32° in medium with BrdU at the indicated concentration. BrdU signal was detected in isolated nuclei. (B) Relative viability during 32.6 µM BrdU incubation, comparing nonincorporating (wt) orhsv-tk+ hENT+ cells, both wild type (wt-inc) and checkpoint mutants. Means ± SEM of three experiments are shown. (C) As in A, time course of EdU incorporation at 1 and 10 µM doses in HU-synchronized cells postrelease. Whole cells were treated with ClickIt reaction before flow cytometry. (D) Relative viability in 10 µM EdU treatment over time for nonincorporating (wt) or incorporating wild-type (wt-inc) or checkpoint-mutant incorporating cells. Means ± SEM of three experiments are shown. (E) Cells were counted during BrdU or EdU treatment to determine proliferation in nonincorporating (wt) orhsv-tk+ hENT+ wild-type (wt-inc) and checkpoint-mutant cells. Cell concentrations were normalized to the 0-hr sample for each cell line/condition and are shown as means ± SEM (n = 3).
Media formulation alters BrdU and EdU sensitivity. (A) Serial dilution assay in YES medium of nonincorporating wild-type (WT) andhsv-tk+ hENT+ WT and checkpoint-mutant strainscds1Δ,chk1Δ,rad3Δ, andmrc1Δ. Plates containing BrdU, thymidine (Thy) control, or EdU were compared using 1/5 dilutions of cells, grown at 32° for 3 days. (B) As in A, spot tests on defined nitrogen-rich EMM medium. Refer toFigure S1 for PMG media effects.
BrdU and EdU cause prolonged DNA synthesis, cell-cycle slowing, and DNA damage. (A) DNA synthesis profiles of wild-type nonincorporating (non-inc) and incorporating (Inc) cells, out of hydroxyurea arrest (HU), released into medium with 32.6 or 326 µM BrdU to detect DNA replication. Left, whole-cell DNA content (SytoxGreen) FACS profiles. Right, septation index for non-inc (NI) and Inc (I) cells stained with aniline blue and DAPI, at different BrdU doses (µM). (B) As in A, cells released from HU were released into medium with 1 or 10 µM EdU. Left, FACS profiles of whole-cell DNA content. Right, septation index at different EdU doses (µM). (C) Asynchronous (AS) cells were treated with 2 mM thymidine (+Thy) or DMSO (vehicle control) for 3 hr (32°) and then released for 0.75 hr. DNA content (SytoxGreen FACS) for each time point, analyzed by FACS, is shown at each point. DMSO control was to test response to DMSO only and was not released. Similar results were seen with aqueous thymidine solution (not shown). (D) Cells were exposed to 32.6 µM BrdU for 2 hr (32°) and then processed for BrdU and phospho-histone H2A (p-H2A) immunofluorescence. DNA was counterstained with DAPI. Merged image is BrdU and p-H2A signals. Bar, 10 µm.
BrdU exposure triggers the DNA damage response. (A) Cells were treated with the indicated dose of BrdU and harvested for protein extraction hourly. Chk1-HA was detected with anti-HA (16B12); the asterisk indicates nonspecific background signal and the open arrow indicates phospho-Chk1HA. Crb2 modification is also indicated by an open arrow. Cds1-myc was detected with anti-myc (solid arrow). Phospho-Cdc2 (p-Cdc2, open arrow) and Cdc2 were also detected. PCNA and β-tubulin are loading controls. The solid line indicates a split between two independent gels, with identical lysates. (B) Incorporating strain viability (FY5031) proportional to BrdU dose is shown as means of three independent experiments ± SEM. (C) Chk-HA and Crb2 phosphorylation after 3 hr EdU (µM), in wild-typehsv-tk+ or nonincorporating cells. BrdU (326 µM) is included as a control. Chk1-HA and Crb2 band shifts are indicated with open arrows. The asterisk indicates nonspecific background band (above Chk1HA) detected using a different antibody from A (αHA, 12CA5). (D) Phospho-Cdc2 (p-Cdc2) after 3 hr BrdU or EdU exposure (doses µM). Below bands are the quantified band intensities of p-Cdc2, normalized to total Cdc2 (below). (E) Quantification of p-Cdc2, relative to total Cdc2 levels, from three independent experiments. Mean values are ± SEM.
BrdU and EdU induce a DNA damage response. (A) Rad52-YFP foci were monitored in untreated cells (untrt) or after 3 hr BrdU at 32°. Rad52 foci (left) or DAPI-stained nuclei (right) are shown on a transmitted light background. Bar, 10 µm. (B) Quantification of three independent experiments in A. Shown are proportions of nuclei with two or more Rad52-YFP foci after 3 hr BrdU ± 95% confidence interval (C.I.). (C and D) Time points selected from movies of wild-type (C; andFile S3 ) orcds1Δ (D; andFile S4 ) cells treated with 10 µM EdU. Arrow (in D) indicates cell that forms foci and lyses. Bar, 10 µm. (E) BrdU incorporated is similar at 32.6 and 326 µM doses. Shown is the mean BrdU signal per dot (±SEM, three independent experiments) at 2.5, 0.25, or 0.025 µg of heat-denatured total DNA, blotted and detected with BrdU antibody. Below, example of BrdU detection on DNA spots. (F) Wild-type nonincorporating orhsv-tk+ hENT+ cells were treated BrdU doses for 3 hr, plated on YES, and then irradiated with 100 J/m2 UV light. Comparison plates were not treated with BrdU, to calculate percentage of viability after BrdU+UV treatment. Shown is the mean viability after BrdU+UV relative to BrdU only, for three independent experiments ± SEM.
BrdU pretreatment changes sensitivity to DNA-damaging drugs. (A–D) Cells were either untreated (untrt) or pretreated (+BrdU) with 32.6 µM BrdU for 2 hr at 32° and then spotted onto drug plates in a 1/5 serial dilution. All plates are YES medium. Arrows, far right, indicate strains that were more sensitive to drug following BrdU pretreatment. Strains FY3454, -2317, -3179, -5148, -5149, and -5150 are shown. Also refer toFigure S3 . (A) YES control for plating efficiency. (B) Sensitivity to phleomycin. (C) Camptothecin (CPT) sensitivity. (D) Hydroxyurea (HU) sensitivity. (E) Forward mutation analysis for loss of Can1 wild-type status. Cells were incubated with 32.6 µM BrdU for 2 hr (32°) and then plated to assess colony number on titer dishes. Remaining culture was plated on PMG + canavanine and incubated 7 days at 32°. Mutation rate, per 107 generations, was calculated comparingcan1− mutants that grew on canavanine plates to the total number plated. Refer to Table 2 for significance results.
Spd1 protects cells from division and mutation during dNTP imbalance. (A) Comparison between wild-type andspd1Δhsv-tk+ hENT+ strains by spot test on EMM plates containing BrdU, EdU, or thymidine. DMSO is a vehicle control for EdU. Shown is the minimal dose where wild-type cells began to show sensitivity to analogs. Strains FY2317, -3454, and -6247 are shown. (B) Cultures were treated with 32.6 µM BrdU and plated to calculate viability relative to 0 hr. Wild-type (wt) andspd1Δ strains expresshsv-tk+ hENT+ (FY2317 and -6247), while the nonincorporating control (non-inc, FY3454) does not. Shown are mean viability values from three independent experiments ± SEM. (C) Proliferation was monitored by counting cell concentration during BrdU treatment for cultures as in A, in addition torad3Δhsv-tk+ hENT+ (FY5150). Shown are mean viability values (n = 3 experiments) ± SEM. (D) Canavanine mutation was scored for incorporating wild-type andspd1Δ strains (FY2317 and -6247), with or without 32.6 µM BrdU treatment (2 hr, 32°). Lea and Coulson fluctuation analysis was used to calculate the rate ofcan1− forward mutation (per 107 generations) in independent cultures over three experiments (wt,n = 12;spd1Δ,n = 15). Shown are median mutation rates with quartile bounding boxes and 95% C.I. error whiskers. Significance was assessed by two-tailed pairwise Mann–WhitneyU-tests, *P = 0.0001, **P < 0.0001. (E) Colonies of wild-type orspd1Δ cells, untreated (no drug) or following 2 hr in 32.6 µM BrdU, were grown on YES and then replicated onto medium with FUdR to score for hsv-tk+ loss. Significance was assessed by two-tailedZ-test (**P < 0.0002). (F) Enhanced sectoring of colonies on FUdR was noted forspd1Δ cells either untreated or following BrdU exposure as in E (two-tailedZ-test, **P < 0.0002), compared to BrdU-treated wild-type cells. Inset, example ofspd1Δ colony with hsv-tk+ loss (*) or sectored area (arrow). Frequencies were calculated from independent experiments, presented with 95% C.I. (G) Model for the effect of exogenous thymidine (Thy) and nucleoside analogs in fission yeast cells expressing a reconstituted thymidine salvage pathway (hsv-tk+). Details are inDiscussion.
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