Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Abstract

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

Keywords: DNA replication; Saccharomyces cerevisiae; cell cycle; flow cytometry; nucleotide excision repair; ultraviolet light.

FIGURE 1.

Mec1 promotes NER-mediated removal of CPD during S phase in S. cerevisiae. A and B, quantification of CPD removal in S. cerevisiae using flow cytometry. WT and rad4Δ cells were synchronized and maintained in G₂/M with nocodazole, mock-treated or irradiated with 100 J/m² UV, and incubated in the dark for various times post-UV. A, FACS DNA content analysis. FACS gating of G₂ cells is indicated with brackets. B, cells were fixed and labeled with anti-CPD (Alexa647) and SYTOX Green. Histograms show the Alexa647 (CPD) fluorescence signal of gated G₂/M cells. C, Mec1 promotes CPD removal during S phase. Cells were synchronized in G₁ with α-factor (α) and released in S with 200 mm HU for 1 h. Synchronized cells were mock-treated or irradiated with 100 J/m² UV, and CPD removal was quantified as in B. Top, FACS DNA content analysis. Bottom, removal of CPDs in S phase. The geometric mean of Alexa647 signals was used to evaluate CPD removal. D, same as C, but cells were released into S phase for 30 min without HU before UV. E and F, Mec1 does not influence CPD removal in G₁ or G₂/M. Samples were treated as in C, except that cells were synchronized and maintained in medium containing α-factor (E) or nocodazole (F). G, quantification of CPD removal in S phase by standard slot-blot assays. Cells were treated as in C. Top, 100 ng of genomic DNA/sample was transferred to nylon membranes by slot-blot and probed with anti-CPD antibodies. Bottom, quantification of signal intensity by densitometry. H, same as G, but cells were maintained in G₁ with α-factor. I, same as G, but cells were maintained in G₂/M with nocodazole. For each panel, values represent the mean ± S.E. (error bars) of three independent experiments.

FIGURE 2.

Rad53, but not Mec1-dependent phosphorylation of Rad26, promotes removal of CPD in S phase. A, removal of CPD in S phase was measured in rad26Δ mutants expressing Myc-RAD26 or Myc-rad26 S27A from pRS316. Top, immunoblot samples were prepared from asynchronously growing cells. Bottom, percentage of CPD remaining in S phase 1 h after UV was measured as in Fig. 1C. B, Rad53 promotes CPD removal during S phase. Cells were synchronized with α-factor and maintained in early S phase using 200 mm HU prior to UV irradiation (100 J/m²). Left, FACS DNA content analysis. Right, removal of CPD as assessed by flow cytometry. C, Rad53 does not influence NER in G₂/M. Cells were treated as in Fig. 1F. For each panel, values are the mean ± S.E. (error bars) of three independent experiments.

FIGURE 3.

S phase defects in removal of CPD induced by MEC1 mutation depend on ongoing DNA replication. WT or mec1Δ sml1Δ cells expressing a thermosensitive cdc7-4 allele were synchronized in G₁ with α-factor and released into S phase for 1 h at the restrictive temperature (37 °C). Cells were then irradiated with UV and incubated at 25 or 37 °C. Removal of CPD was measured by flow cytometry. Values are the mean ± S.E. (error bars) of three independent experiments.

FIGURE 4.

Mutations in cellular pathways involved in the response to replicative stress cause NER defects during S phase. A and B, cells lacking the histone H3 Lys-56 acetyltransferase Rtt109 present NER defects specifically in S phase. A, removal of CPD was measured in S phase by flow cytometry. B, removal of CPD was measured in G₂/M by flow cytometry. C, percentage of CPD remaining in S phase at 1 h post-UV irradiation (100 J/m²) was assessed as in Fig. 1C in the indicated mutant strains. Strains presenting significantly slower removal of CPD compared with WT are shown in dark gray (p < 0.01, two-tailed unpaired t test), whereas those displaying no significant defect are shown in light gray. D, cells were synchronized in G₂/M with nocodazole or in S phase without HU as in Fig. 1D. Serial dilutions were spotted on YPD-agar, and plates were mock-treated or irradiated with UV (25 J/m²). E, cells were synchronized in G₁ with α-factor. UV irradiation (100 J/m²) was performed either in G₁, in early S phase after release in 200 mm HU, in mid-S phase after release without HU, or after release into nocodazole for 2 or 4 h, respectively. Left, FACS DNA content analysis. Right, percentage of CPD remaining at 1 h post-UV irradiation (assessed by flow cytometry). For each panel, values are the mean ± S.E. (error bars) of three independent experiments.

FIGURE 5.

Mutants defective in NER during S phase present elevated frequencies of spontaneous and genotoxin-induced Rfa1 foci. A, Rfa1-GFP-expressing cells of the indicated genotype were grown to the exponential phase, fixed with formaldehyde, and examined by fluorescence microscopy. Strains are grouped as being S phase repair-proficient (SPR+) or -deficient (SPR−). Left, the percentage of cells containing visible foci was counted in the total cell population (G₁, S, and G₂/M cells) or in small-budded cells only (S phase). Representative images of cells in each cell cycle phase are shown. Middle, CPD removal of the SPR+ and SPR− groups is presented as a scatter plot (data from Fig. 4C). Right, percentage of foci-positive cells in each group presented as a scatter plot. *, p < 0.01, two-tailed unpaired t test between SPR+ and SPR−. B, effect of HU on Rfa1-GFP foci formation. Rfa1-GFP-expressing cells were synchronized in G₁ and released toward S phase for 1 h with or without 200 mm HU. Left, cell cycle profiles before and after release (WT cells), with typical pictures of budded shmoos (cells that entered S phase) at 1 h. Right, budded shmoos with Rfa1-GFP foci at 1 h were counted as in A. C, Rfa1-YFP cells were synchronized in G₁ and released toward S phase in medium containing 200 mm HU for 1, 2, or 4 h. S phase cells (budded shmoos as in B) with Rfa1-YFP foci were counted at each time point. D, cells were synchronized and treated with HU as in C. After the indicated time period in HU, cells were irradiated with UV (100 J/m²), and the percentage of CPD remaining 1 h post-UV was measured by flow cytometry. E, effect of 4NQO on Rfa1-GFP foci formation. SPR− and SPR+ strains were arrested in G₁ and released for 2 h in medium containing 150 ng/ml 4NQO. Left, cell cycle profile before and after 4NQO treatment of WT Rfa1-GFP cells, with typical pictures of cells at each time point. Right, cells with Rfa1-GFP foci were counted as in A at 0 h (G₁ shmoos) and 2 h (budded shmoos). *, p < 0.001, two-tailed unpaired t test between SPR+ and SPR−. F, effect of acute MMS treatment on the induction of Rfa1-YFP foci. G₁-arrested cells were released for 1 h in 0.025% MMS. After inactivation of MMS with 2.5% thiosulfate, cells were further incubated in YPD. Samples were collected at the indicated times, and the fraction of cells with Rfa1-YFP foci was determined as in A. *, p < 0.01, χ² between WT and mutants for each time point. For each panel, at least 200 cells were counted per sample. Experiments were repeated twice, and representative data are shown. Error bars, S.E.

FIGURE 6.

Partial depletion of Rfa1 causes S phase-specific defects in CPD removal. A, exponentially growing cells expressing Tir1-Myc alone or in combination with Rfa1-AID-6FLAG were incubated in YPD medium containing 2 mm auxin for 1 h. Samples were then analyzed by immunoblotting. B, cells were spotted on YPD-agar containing the indicated concentrations of auxin. C, cells were synchronized in S phase with HU (left) or G₂/M with nocodazole (right) as in Fig. 1, except that 0.5 mm auxin was added to cultures 30 min prior to UV irradiation. Levels of Rfa1-AID-6FLAG were monitored by immunoblot (top). Removal of CPD following UV irradiation was measured by flow cytometry. D, effect of partial RPA depletion on S phase progression. Tir1-Myc Rfa1-AID-6FLAG cells were arrested in G₁ and were then pretreated for 30 min with or without 0.5 mm auxin before release at room temperature with or without auxin. Samples were collected at 5-min intervals for FACS cell cycle analysis. Left, cell cycle profiles for representative time points. Right, the average DNA content in each sample relative to G₁ is used as a measure of S phase progression. For each panel in this figure, values represent the mean ± S.E. (error bars) of three independent experiments.

FIGURE 7.

Defects in S phase NER are rescued by RPA overexpression in human cells. RPA was overexpressed in human cells using a polycistronic vector in which each RPA subunit is separated by self-cleaving P2A peptide (pAC-GFP-C1-RPA3-P2A-RPA1-P2A-RPA2). This results in stoichiometric overexpression of subunits of the RPA complex, with RPA3 being fused to GFP. A GFP expression vector was used as negative control (empty vector). A and B, overexpression of RPA in HeLa cells rescues S phase NER defects induced by pharmacological ATR inhibition. Transfected cells were pretreated for 2 h with a 10 μm concentration of the ATR inhibitor VE-821, followed by irradiation with 20 J/m² UV or mock irradiation. A, total protein extracts were prepared 1 h after UV irradiation and analyzed by immunoblot. RPA1 and RPA2 resulting from P2A cleavage (RPA1-P2A and RPA2-P2A) have a slightly higher molecular weight compared with endogenous counterparts. B, HeLa cells were treated as in A, and removal of UV-induced 6-4PP was measured as a function of cell cycle using a flow cytometry-based assay (see “Experimental Procedures”). C, HeLa cells were treated with VE-821 and mock- or UV-irradiated, as in A and B. Recruitment of RPA2 to chromatin after UV was detected by immunofluorescence microscopy. BrdU labeling was used to identify S phase cells. D, the average intensity of fluorescence for RPA2 signal was quantified for each condition described in C (at least 400 cells/condition). Values are presented as box plots (median, first, and third quartile, with the whiskers representing the first and last 5%). *, p < 0.0001, two-tailed unpaired t test. E, XP30ROsv, an SV40-transformed skin fibroblast derived from an XPV patient, was transfected with the polycistronic RPA expression vector or control GFP expression vector. Removal of 6-4PP following UV was measured as in B. An isogenic clone expressing polη (CL6) was used as a control. F, recruitment of RPA to chromatin after UV was measured in XP30ROsv and CL6 as described in D. *, p < 0.0001, two-tailed unpaired t test. G, U2OS osteosarcoma and WM3248 human melanoma cell lines were transfected with the polycistronic RPA expression vector or control vector. Removal of 6-4PP following UV was measured as in B. H, effect of RPA overexpression on rates of DNA synthesis. HeLa cells were transfected with RPA or control vector and incubated with 30 μm BrdU for 0, 30, 60, and 90 min. Cells were labeled with an anti-BrdU-Alexa647 antibody and PI and analyzed by flow cytometry. Top left, example of bivariate plot after 60-min labeling. Black rectangle, gate used to select S phase cells. Top right, the geometric mean of BrdU signal in S phase cells was used to calculate the incorporation of BrdU in DNA as a function of time. Bottom panels, representative histograms of BrdU signal of gated S phase population. For flow cytometry data in this figure, values represent the mean ± S.E. (error bars) of three independent experiments. Microscopy data combines values from two independent experiments.