Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Abstract

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.

Fig. 1.

Accumulation of hEXO1a and hEXO1b at LUDs. (A and B) MRC5VI cells were transfected with hEXO1a-mCherry (A) or hEXO1b-mCherry (B), seeded on coverslips, and locally UV irradiated (40 J/m²) through Isopore filters. After an additional 1-h incubation, cells were fixed and processed for indirect immunofluorescence using Abs against XPA, XPB, and RPA (Alexa Fluor 488), and nuclei were counterstained with DAPI. White arrows indicate LUDs. (Scale bar: 5 μm.) (C) Quantification of immunofluorescence shown in A and B. Histograms represent the mean ± SD of three independent experiments. Approximately 40 LUDs were scored for each experiment. (D) MRC5VI cells were mock- or UV-treated and harvested; lysates were prepared and resolved directly on SDS/PAGE (WCE, whole cell extracts, 10%) and incubated with preimmune serum (PI) as a control or anti-hEXO1 Ab (F15). Western blot analysis was performed with hEXO1 Ab (Ab4) and XPA Ab (12F5). XPA runs as a doublet because of incomplete reduction (32). S, immunoprecipitation flow-through; I, immunoprecipitate.

Fig. 2.

NER preincision complex and 5′ incision are required for hEXO1b accumulation at LUDs. (A) MRC5VI, XP12RO (XPA), XP2YO (XPF), XP2YO (XPF+XPF D676A), XPCS1RO (XPG), and XPCS1RO (XPG+XPG E791A) cells were transfected with hEXO1b-mCherry, seeded on coverslips, and locally UV-irradiated (40 J/m²) through Isopore filters. After 1 h of incubation, cells were fixed and processed for immunofluorescence against the indicated protein and nuclei were counterstained with DAPI. White arrows indicate the position of LUDs. (Scale bar: 5 μm.) (B) Quantification of immunofluorescence shown in A. Histograms show the mean ± SD of three independent experiments. Approximately 40 LUDs were scored for each experiment.

Fig. 3.

Accumulation of hEXO1b at LUDs is enhanced when repair synthesis is blocked. MRC5VI and XP12RO (XPA) cells transfected with hEXO1b-mCherry were exposed to local UV irradiation (40 J/m²) and incubated for 1 h at 37 °C in the presence or absence of Ara-C. (A) Histograms indicating the percentage of XPB-positive LUDs that also contained hEXO1b-mCherry, with or without Ara-C treatment. Approximately 40 LUDs were scored in each of three independent experiments; values are mean ± SD. (B) Representative images of cells with hEXO1b-positive LUDs (with or without Ara-C treatment) in the MRC5VI control (Upper) and in the XPA mutated cell lines (Lower). LUDs are indicated by white arrows. (Scale bar: 5 μm.)

Fig. 4.

Down-regulation of hEXO1 impairs UDS after UV irradiation. (A) Primary 48BR cells were depleted of hEXO1, XPA, or control (LUC); cultured on coverslips; and UVC-irradiated (20 J/m²), followed by incubation with 10 μM EdU for 3 h, fixation, and conjugation of the fluorescent dye to incorporated EdU. The intensity of nuclear fluorescence, which is associated with UDS activity, was measured using a fluorescence microscope and image-processing software. For each sample, at least 100 nuclei were analyzed in three independent experiments; error bars represent SD. (B) Silencing efficiency was monitored by Western blot analysis for XPA (Upper) and by RT-PCR for hEXO1 (Lower).

Fig. 5.

hEXO1 modulates a crucial step linking UV-induced lesions to histone H2A ubiquitylation. (A) Quiescent 48BR cells were transfected with siRNA directed against luciferase or hEXO1 (two different sequences, sihEXO1-1 and sihEXO1-2, were used) and locally UV-irradiated. At 1 h after UV irradiation, cells were processed to detect uH2A and XPA by immunofluorescence. Images show one representative nucleus for each treatment. (Scale bar: 5 μm.) (B) Quantification of the percentage (mean ± SD) of XPA-positive LUDs that are also positive for uH2A in siLUC, sihEXO1-1, and sihEXO1-2. (C) Silencing efficiency was monitored by RT-PCR.

Fig. 6.

hEXO1 is required for checkpoint activation after UV irradiation in quiescent human primary fibroblasts. Quiescent human primary fibroblasts transfected with siRNA against hEXO1 (sihEXO1-1 was used) or XPA were held under low serum conditions for 3 d before mock or UV irradiation. (A) Activation of the DNA damage checkpoint was examined at 1 h after UV irradiation by Western blot analysis using anti-P-Ser-317-Chk1. (B) Cells were UV-irradiated (20 J/m²) and harvested at different time points (1 or 2 h) after treatment. The DNA damage checkpoint was monitored by Western blot analysis using anti–P-Ser-15-p53 Ab. (C) hEXO1 knockdown was analyzed by RT-PCR.