PARP2 controls double-strand break repair pathway choice by limiting 53BP1 accumulation at DNA damage sites and promoting end-resection

Abstract

Double strand breaks (DSBs) are one of the most toxic lesions to cells. DSB repair by the canonical non-homologous end-joining (C-EJ) pathway involves minor, if any, processing of the broken DNA-ends, whereas the initiation of DNA resection channels the broken-ends toward DNA repair pathways using various lengths of homology. Mechanisms that control the resection initiation are thus central to the regulation to the choice of DSB repair pathway. Therefore, understanding the mechanisms which regulate the initiation of DNA end-resection is of prime importance. Our findings reveal that poly(ADP-ribose) polymerase 2 (PARP2) is involved in DSBR pathway choice independently of its PAR synthesis activity. We show that PARP2 favors repair by homologous recombination (HR), single strand annealing (SSA) and alternative-end joining (A-EJ) rather than the C-EJ pathway and increases the deletion sizes at A-EJ junctions. We demonstrate that PARP2 specifically limits the accumulation of the resection barrier factor 53BP1 at DNA damage sites, allowing efficient CtIP-dependent DNA end-resection. Collectively, we have identified a new PARP2 function, independent of its PAR synthesis activity, which directs DSBs toward resection-dependent repair pathways.

Figure 1.

PARP2 depletion alters DSB repair efficiency. (A) Effect of PARP2 stable depletion on X-rays induced cytotoxicity measured using a clonogenic surviving assay. The analysis was performed in control SV40-immortalized human fibroblast (HF shcontrol) and in the PARP2 stably depleted cells (HF shPARP2). Each treatment was repeated three times in quadruplicate. Data represents the mean ± SEM. **P < 0.01; ***P < 0.001 (Mann-Whitney test). (B) Clonogenic cell survival of HF shControl or HF shPARP2 cells in response to bleomycin. Cells in suspension were exposed to the indicated bleomycin concentration for 1 h before plating in drug-free media. Each treatment was repeated four times in triplicate. Data represents the mean ± SEM. **P < 0.01; ***P < 0.001 (Mann–Whitney test). The expression level of the PARP2 protein in the different cell cultures was assessed by western blotting. (C) Western blot analysis of whole cell extracts from HF EJ-CD4 cells transfected with the indicated siRNA. Cells extracts were prepared 48 h post siRNA transfection, corresponding to the I-SceI transfection time point. The relative repair efficiency by HR of I-SceI-induced double strand cut is decreased when PARP2 is depleted in (D) HF DR-GFP cells and (E) U2OS DR-GFP cells. (F) The relative repair efficiency by EJ of I-SceI-induced double strand cut is increased when PARP2 is depleted in HF EJ-CD4 cells. Values plotted represent relative repair efficiency calculated as a ratio of repair efficiency measured in cells transfected with siControl. Values are mean ± SEM from five (HF DR-GFP cells), six (U2OS DR-GFP cells) and five (in HF EJ-CD4 cells) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

PARP2 promotes HR and A-EJ independently of its PARylation activity. HF DR-GFP cells (A) and HF EJ-CD4 cells (B) were transfected with the indicated siRNA for two days. When indicated, the PARP inhibitor Veliparib (10 μM) was added to the cell culture medium 1 h before transfection with the DsRed-I-SceI expression plasmid and kept until the relative repair efficiency was analysed. Data represent mean ± SEM from five experiments. **P < 0.01; ***P < 0.001; ns, not significant. (C) Clonogenic cell survival was measured in HF EJ-CD4 cells transfected with the indicated siRNA and complemented with the wild type GFP-PARP2 or the PAR synthesis dead mutant GFP-PARP2 E545A. Cells in suspension were exposed to the indicated bleomycin concentration for 1 h before plating in drug-free media. Each treatment was repeated four times in triplicate. Data represents the mean ± SEM. *P < 0.05; ns, not significant. (D) The EJ repair efficiency was measured in HF EJ-CD4 cells transfected with the indicated siRNA and complemented with the wild type GFP-PARP2 or the PARylation dead mutant GFP-PARP2 E545A. Data represent mean ± SEM from three experiments, **P < 0.01; ***P < 0.001. (E) PARP2 depletion lowers the A-EJ repair efficiency. Analysis of the DNA sequence of repair junction from HF EJ-CD4 cells depleted for PARP1 or PARP2 and complemented or not with the wild type GFP-PARP2 or the PAR synthesis dead mutant GFP-PARP2 E545A. The EJ repair events are categorized according to the DNA sequence found at the repair junction. The events limited to the 3′Pnt are repair events for which the sequence at the repair junction includes at least one of the four nucleotides from the 3′Pnt generated by I-SceI cleavage; these events are dependent on the C-EJ pathway. The events with deletion exceeding the 3′Pnt, are events for which the sequence at the repair junction have four or more of the nucleotides at the I-SceI cleavage site that have been deleted; these events are dependent on the A-EJ pathway. Numbers indicate the percentage of junction events of either type and result from the analysis of 36–147 junction sequences per condition. (F) The graph represents the percentage of events with deletion of more than 20 base pairs at the repair junction. *P < 0.05, statistics are presented only when differences are significant.

Figure 3.

PARP2 stimulates DNA end-resection. (A) Cell transfected for 48 h with the indicated siRNA were then complemented with the wild type GFP-PARP2 or the PAR synthesis dead mutant GFP-PARP2 E545A and treated or not for 2 h with 25 μg/ml bleomycin and released into drug-free medium for 6 h (6 hours release). The cells were fixed and stained for the immunodetection of RAD51. Data represent mean number of foci per nuclei ± 95% CI from at least 850 cells analysed from three independent experiments. ***P < 0.001 (one-way ANOVA test). (B) Western blot analysis of whole cell extracts from U2OS cells transfected with the indicated siRNA. (C) The proportion of U2OS cells positive for RPA32 subunit signal is presented in siControl and siPARP2 transfected cells. Cell transfected for 48 h with the indicated siRNA were untreated (UT) or treated for 2 h with 25 μg/ml bleomycin and then released in fresh medium for the indicated time points (0 h, indicates no release). The soluble proteins were extracted and the cells were stained for the immunodetection of the RPA32 subunit and analysed by flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (D) Representative flow cytometry dot plot graphs showing the U2OS cell population treated as indicated and stained with propidium iodide (PI) and for RPA32 (RPA). (E) Analysis of the number of BrdU foci in siControl and siPARP2 transfected U2OS cells. Cells were transfected with the indicated siRNA for 48 h were treated or not for 2 h with 25 μg/ml bleomycin and released into drug-free medium for 6 h (bleomycin release time, 6 h). Cells were then processed for BrdU immunofluorescence without DNA denaturation at the indicated time post-release. Data represent mean number of foci per nuclei ± 95% CI from a total of at least 850 cells analysed from three independent experiments. *P < 0.05; ns, not significant (one-way ANOVA test). (F) Representative images of U2OS cells fixed on coverslips and BrdU immunostained for foci quantification presented in (E).

Figure 4.

PARP2 stimulates DNA end-resection by preventing 53BP1 accumulation at broken DNA ends. (A) U2OS cells were transfected for 48 h with the indicated siRNA and when indicated the siRNA transfected cells were complemented with the wild type GFP-PARP2 or the PAR synthesis dead mutant GFP-PARP2 E545A. The cells were treated or not for 2 h with 25 μg/ml bleomycin and were released into drug-free medium for 6 h (6 h release). The cells were fixed and stained for the immunodetection of 53BP1. Data represent the mean number of foci per nuclei ± 95% CI from at least 1200 cells analysed from three independent experiments. ***P < 0.001 (one-way ANOVA test). (B) Representative images of U2OS cells fixed on coverslips, and 53BP1 immunostained for foci quantification presented in (A). (C) HeLa cells were transfected with siControl or siPARP2 for 48 h and were subsequently transected for 24 h with GFP-53BP1 fusion protein expression plasmid. For the recruitment analysis, 10 μg/ml Hoechst 33258 was added to the culture medium 5 min before induction of DNA damage by micro-irradiation with a two-photon laser (810 nm). The irradiated cells were imaged every 10 s for 10 min. Data represents mean relative spot intensity ± SEM, n = 25–28 individual cells from a minimum of five independent experiments. (D) Representative images of the recruitment of the GFP-53BP1 fluorescent-tagged protein. (E) The relative repair efficiency by HR is determined in HF DR-GFP cells transfected with the indicated siRNA. Data represent mean ± SEM from four experiments. * P < 0.05, statistics are presented only when differences are significant. (F) The relative repair efficiency by SSA is determined in U2OS SA-GFP cells transfected with the indicated siRNA. Data represent mean ± SEM from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (G) The relative repair efficiency by EJ was measured in HF EJ-CD4 cells transfected with the indicated siRNA. Data represent mean ± SEM from at least three experiments. *P < 0.05; ***P < 0.001, statistics are presented only when differences are significant. (H) Analysis of the DNA sequence of repair junction from HF EJ-CD4 cells transfected with the indicated siRNA. Data represent the percentage of repair events of each class and result from the analysis of 41–147 junction sequences per condition. (I) Analysis of proteins expression by western blot of cell extracts from HF EJ-CD4 cells from (G) and (H).

Figure 5.

CtIP is required for PARP2 to stimulate DNA end-resection. Analysis of (A) CtIP or (B) BRCA1 protein expression by western blot of whole cell extracts prepared from HF EJ-CD4 cells transfected with the indicated siRNA. Protein extracts prepared 48 h after transfection with the indicated siRNA. The relative repair efficiency of I-SceI-induced double strand ends by (C) HR in HF DR-GFP cells or by (D) SSA in U2OS cells carrying the SA-GFP substrate was determined. Values are mean ± SEM from at least four (HF DR-GFP) and least three (U2OS SA-GFP) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (E) The relative repair efficiency of I-SceI-induced double strand ends by EJ in the HF EJ-CD4 cells transfected with the indicated siRNA was determined. Data represent mean ± SEM from at least three experiments. *P < 0.05; **P < 0.01; ns, not significant. (F) Analysis of the DNA sequence of repair junction from HF EJ-CD4 cells transfected with the indicated siRNA. Data represent the percentage of repair events of each classes of EJ events and result from the analysis of 26–147 junction sequences per condition. (G) Effect of PARP2 and/or BRCA1 depletion on bleomycin induced HeLa cells cytotoxicity assessed using a clonogenic surviving assay. The HeLa shControl and HeLa shBRCA1 were transfected with the indicated siRNA for 48 h. Cells in suspension were exposed to the indicated bleomycin concentration for 1 h before plating. Each data point represents mean ± SEM of six independent experiments with each dose in triplicate. **P < 0.01; ***P < 0.001, statistics are presented only when differences are significant (Mann-Whitney test). (I) Representative immunoblot showing the levels of the indicated proteins HeLa cells 48 h post transfection with the indicated siRNA.

Figure 6.

Model for the role of PARP2 in the DSBR pathway choice.