XPA-mediated regulation of global nucleotide excision repair by ATR Is p53-dependent and occurs primarily in S-phase

Abstract

Cell cycle checkpoints play an important role in regulation of DNA repair pathways. However, how the regulation occurs throughout the cell cycle remains largely unknown. Here we demonstrate that nucleotide excision repair (NER) is regulated by the ATR/p53 checkpoint via modulation of XPA nuclear import and that this regulation occurs in a cell cycle-dependent manner. We show that depletion of p53 abrogated the UV-induced nuclear translocation of XPA, while silencing of Chk1 or MAPKAP Kinase-2 (MK2) had no effect. Inhibition of p53 transcriptional activities and silencing of p53-Ser15 phosphorylation also reduced the damage-induced XPA nuclear import. Furthermore, in G1-phase cells the majority of XPA remained in the cytoplasm even after UV treatment. By contrast, while most of the XPA in S-phase cells was initially located in the cytoplasm before DNA damage, UV irradiation stimulated bulk import of XPA into the nucleus. Interestingly, the majority of XPA molecules always were located in the nucleus in G2-phase cells no matter whether the DNA was damaged or not. Consistently, the UV-induced Ser15 phosphorylation of p53 occurred mainly in S-phase cells, and removal of cyclobutane pyrimidine dimers (CPDs) was much more efficient in S-phase cells than in G1-phase cells. Our results suggest that upon DNA damage in S phase, NER could be regulated by the ATR/p53-dependent checkpoint via modulation of the XPA nuclear import process. In contrast, the nuclear import of XPA in G(1) or G(2) phase appears to be largely independent of DNA damage and p53.

Figure 1. p53 is required for the XPA nuclear import upon UV irradiation.

A, p53 was transiently knocked down with siRNA duplexes in HeLa cells. After treatment with or without 20 J/m² UV followed by a 2-hr recovery, subcellular fractionation and Western blotting were performed to assess the re-distribution of XPA. β-actin and PARP were probed as cytoplasmic and nuclear protein controls, respectively. The quantitative data were obtained from at least three independent experiments. nXPA/cXPA represents the ratio of nuclear XPA to cytoplasmic XPA. B, Immunofluorescence microscopic analysis of cells transfected with control or p53 siRNA and with or without UV irradiation. C, A549/LXSN(p53+) and A549/E6(p53−) cells were mock- or UV-irradiated. Cytosol and nuclear fractions were collected and analyzed by Western blotting. D, A459 cells were pre-treated with pifithrin-α (30 uM), an inhibitor of p53 transcriptional activity, for 20 hrs. After UV irradiation and a 2-hr recovery, the cells were analyzed for subcellular localization of XPA. The * in the plots indicates a statistically significant (p<0.05) difference between the groups being compared.

Figure 2. Cell cycle checkpoint proteins Chk1 and MK2 are not required in the UV-induced nuclear import of XPA.

A. siRNA duplexes targeting Chk1 were transiently transfected into A549 cells, followed by mock or 20 J/m² UV irradiation and a 2-hr recovery. The localization of XPA was assessed using subcellular fractionation followed by Western blot analysis. PARP and β-actin proteins were probed as nuclear and cytoplasmic protein controls, respectively. B. A549 cells were treated with MK2 or control siRNA, followed by UV irradiation. After a 2-hr recovery period, irradiated cells were fractioned and analyzed by Western blotting. C. Chk1 and MK2 were simultaneously knocked down by Chk1 and MK2 siRNAs, or ATR was knocked down by ATR siRNA. Then, the UV-induced XPA nuclear import in these cells was assessed by fractionation and Western blotting.

Figure 3. DNA damage-induced XPA nuclear accumulation occurs primarily in S-phase.

A. Mitotically-synchronized A549 cells grown for the indicated time periods were stained with propidium iodide for analysis of the cell cycle distribution (Panel A, left) or labeled with BrdU to identify synchronized S-phase cells (panel A, right). B. Immunofluorescence microscopic analysis of the subcellular localization of XPA in the synchronized cells. Synchronized A549 cells were mock- or UV-treated (20 J/m²) and left to recover for 2 hrs. Cells were fixed and stained with primary and fluorescence–conjugated secondary antibodies to determine the localization of XPA. At least 100 cells were examined, and the representative data is shown. C. Left: Mitotically-synchronized HeLa cells were stained with propidium iodide followed by flow cytometric analysis. Right: Subcellular fractionation followed by Western blotting was performed to analyze the subcellular localization of XPA in each phase of the cell cycle after UV irradiation of synchronized HeLa cells. PARP and β-actin were probed as nuclear and cytoplasmic protein controls, respectively. At least three independent experiments were performed and representative data is presented.

Figure 4. Removal of UV-induced DNA damage in G1- and S-phase cells.

A. Mitotically-synchronized HeLa cells were fixed and stained with propidium iodide at the indicated time points following the mitotic “shake off”. The cell cycle distribution then was analyzed by flow cytometry. Cells at G1 (at the 6 hours post-“shake off”) or S (20 hours post-“shake off”) phase, were UV irradiated at 10 J/m², followed by a recovery of 24 hours. B. Cells at G1 or S phase were UV irradiated at 10 J/m², followed by the indicated periods of repair. Cellular DNA were isolated and the removal of CPDs and 6-4PPs was measured by slot-blot assay. The amounts of CPDs or 6-4PPs were normalized to the values at zero hour and quantified based on three independent measurements.

Figure 5. Phosphorylation of p53 is required for the UV-induced XPA nuclear import.

A. Mitotically synchronized A549 cells were mock-treated or irradiated with 20 J/m² of UV-C, and allowed a 2-hr recovery before accessing the phosphorylation of p53 at Ser15 by Western blotting. B. Constructs for expressing human wild-type p53 or the S15A mutant of p53 were co-transfected with p53 3′-UTR siRNA into A549 cells. 72 hours after transfection, the A549 cells were mock- or UV (20 J/m²)-treated and allowed a 2-hr recovery. The UV-induced phosphorylation of p53 and the XPA in the nuclear fraction then were analyzed by Western blotting. The right panel shows the efficiency of siRNA knockdown of endogenous p53 and the level of recombinant p53 in the cells co-transfected with p53 3′-UTR siRNA and p53-WT constructs.