Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin - PubMed (original) (raw)

Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin

Hana Hanzlikova et al. Nucleic Acids Res. 2017.

Abstract

A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established 'isogenic' human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

Development of PARP1-/-, PARP2-/-, PARP3-/- and XRCC1-/- RPE-1 cells and XRCC1 high-content imaging. Wild type (WT), PARP1-/-, PARP2-/-, PARP3-/- and XRCC1-/- RPE-1 clonal cell lines were analysed for loss of the targeted protein by (A) Western blotting and (B) immunofluorescence. Note that the PARP3 antibody available to us was not suitable for immunofluorescence. (C) Left, representative ScanR images of WT and XRCC1-/- RPE-1 cells non-treated or treated with 1 mM hydrogen peroxide (H2O2) for 10 min and pre-extracted with detergent prior to fixation and immunostaining for XRCC1 (green), the nucleolar marker B23 (red) and counterstaining with DAPI (blue). Right, quantification of detergent-insoluble anti-XRCC1 signal (excluding nucleolar XRCC1 signal) from >1000 cells per sample using Olympus ScanR analysis software. Data are the mean (±SEM) of three independent experiments. The black dotted line denotes non-specific anti-XRCC1 background signal, defined as the residual signal in XRCC1-/- RPE-1 cells.

Figure 2.

Figure 2.

Levels of H2O2-induced ADP-ribosylation and XRCC1 recruitment into chromatin in PARP-deleted RPE-1 cells. (A) Levels of the indicated proteins (left) and ADP-ribosylated proteins (right) were compared in cell lysates from the indicated WT or mutant RPE-1 cells harvested before and after treatment with 400 µM H2O2 for 7 min by Western blotting using appropriate antibodies and anti-pan-ADP-ribose binding reagent. (B) Levels of ADP-ribosylation and chromatin-bound XRCC1 were analysed by indirect immunofluorescence in cells treated or not with H2O2 (as above) by fixation and staining with anti-pan-ADP-ribose binding reagent (top panels) or by detergent pre-extraction prior to fixation and staining with anti-XRCC1 antibody (bottom panels). Representative ScanR images are shown.

Figure 3.

Figure 3.

Residual recruitment of endogenous XRCC1 into oxidized chromatin in PARP1-/- RPE-1 cells is greatly reduced by PARP inhibitor. (A) WT and PARP1-/- RPE-1 cells were pre-incubated or not with 10 μM KU0058948 inhibitor for 1 h prior to a 7 min incubation with or without 400 μM H2O2. Cells were pre-extracted with detergent to remove non-chromatin bound proteins prior to fixation and immunostaining with the indicated antibodies or anti-pan-ADP-ribose binding reagent. Representative ScanR images are shown. (B) Quantification of total nuclear pan-ADP-ribose and chromatin-bound nuclear XRCC1 (excluding nucleolar XRCC1 signal) in cells treated as in panel A. Nucleoli were located using anti-B23 antibodies. All data are the mean (±SEM) of three independent experiments with >1000 cells scored per sample in each experiment. Statistical significance was assessed by two-tailed t-tests. Asterisks ** and *** indicate _P_-values of <0.01 and <0.001, respectively; ns – not significant. The black dotted line denotes non-specific anti-XRCC1 background signal, defined as the residual signal in XRCC1-/- cells stained in parallel.

Figure 4.

Figure 4.

Overlapping roles for PARP1 and PARP2 in recruiting endogenous XRCC1 into oxidized chromatin. (A) Levels of ADP-ribosylation and chromatin-bound XRCC1 were measured by indirect immunofluorescence in WT and _PARP1-/-/PARP2-/-_cells treated or not with 400 μM H2O2 for 7 min by fixation and staining with anti-pan-ADP-ribose binding reagent and DAPI (top panels) or by detergent pre-extraction prior to fixation and staining with anti-XRCC1 and anti-B23 antibodies (bottom panels). Representative ScanR images are shown. (B) Quantification of total nuclear pan-ADP-ribose and chromatin bound XRCC1 (excluding nucleolar signal). The black dotted line denotes non-specific anti-XRCC1 background signal, measured by XRCC1 immunostaining in XRCC1-/- cells in parallel. All data are the mean (±SEM) of three independent experiments with >1000 cells scored per sample in each experiment. Statistical significance was assessed by two tailed t-tests. Asterisks * and ** indicate _P_-values of <0.05 and <0.01, respectively; ns – not significant. (C) DNA strand breakage was quantified by alkaline comet assays in indicated RPE-1 cells before, immediately after treatment with 50 μM H2O2 on ice and after the depicted repair periods in drug-free medium. Data are the average comet tail moment (an arbitrary unit-measure of DNA strand breaks) of 100 cells per sample and are the mean (±SEM) of three independent experiments. Statistically significant differences (two-way ANOVA) are indicated (**P < 0.01; ***P < 0.001; ****P < 0.0001; ns – not significant).

Figure 5.

Figure 5.

Overlapping roles for PARP1 and PARP2 in recruiting endogenous PNKP into oxidised chromatin. (A) Levels of chromatin-bound PNKP were analysed by indirect immunofluorescence in indicated RPE-1 cell lines untreated or treated with 400 μM H2O2 for 7 min by detergent pre-extraction prior to fixation and staining with anti-PNKP antibody. Representative ScanR images are shown. (B) Quantification of chromatin-bound PNKP in cells measured as above. All data are the mean (±SEM) of three independent experiments with >2000 cells scored per sample in each experiment. Statistical significance was assessed by two-tailed t-tests. Asterisks * and ** indicate _P_-values of <0.05 and <0.01, respectively; ns – not significant. (C) Levels of PNKP and the relevant proteins in cell extracts from RPE-1 cells of the indicated genotype.

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