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, 34 (21), 6170-82

PARP-1 and Ku Compete for Repair of DNA Double Strand Breaks by Distinct NHEJ Pathways

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PARP-1 and Ku Compete for Repair of DNA Double Strand Breaks by Distinct NHEJ Pathways

Minli Wang et al. Nucleic Acids Res.

Abstract

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.

Figures

Figure 1
Figure 1
Effect on DNA DSB rejoining of PARP-1 inhibitors in LIG4−/− MEFs, as well as of PARP-1 DBD expression in Chinese hamster cells after exposure to IR. (A) p53−/−/LIG4−/− and p53−/− MEFs (wild-type) were pretreated with 10 mM 3′-AB or solvent (DMSO <0.4% v/v in medium) for 1 h, exposed to 30 Gy X-rays and returned to 37°C for repair. At various times thereafter cells were collected and prepared for AFIGE to measure residual DNA DSBs. The upper panel shows gels from a typical experiment and the lower panel the results of their quantitative analysis. Plotted is FDR, a measure of DNA DSBs present, as a function of the repair time. Shown are the means and standard errors calculated from four determinations in two experiments. The value of FDR measured in non-irradiated cells has been subtracted from the results shown. Standard lysis conditions were used in these experiments but similar results have been obtained with low temperature lysis. (B) COM3 cells expressing the PARP-1 DBD under the control of the mouse mammary virus LTR that is dexamethasone inducible were incubated with 50 nM dexamethasone for 24 h or left untreated. Thereafter, cells were exposed to 40 Gy and allowed to repair at 37°C in the presence (20 μM) or absence of wortmannin (given 1 h before IR). Repair of DSBs was measured by AFIGE and is expressed as FDR versus repair time. The upper panel shows typical gels and the lower panel the repair kinetics plotted as FDR versus time. Shown are the means and standard errors calculated from six determinations in three experiments.
Figure 2
Figure 2
Effect of PARP-1 inhibitors on Ku deficient cells. (A) The effect of DPQ (10 μM), or DMSO on poly(ADP-ribose) levels in Ku80 deficient xrs-6 cells, as well as in their corrected counterpart xrs-6/KU80 cells. Cells growing on coverslips were exposed to 40 Gy X-rays and prepared for immunofluorescence 15 min later. The poly(ADP-ribose)-associated fluorescence in non-irradiated, irradiated, as well as irradiated and treated cells is shown for xrs-6 and xrs-6/Ku80 cells, respectively. DPQ inhibits PARP-1 activity in both cell lines. (B) Rejoining of IR-induced DNA DSBs in xrs-6 cells and xrs-6/Ku80 cells in the presence or absence fo DPQ (10 μM). Results from four determinations in two experiments are shown. Other details as in Figure 1. (C) DNA DSB repair kinetics in Ku70 deficient cells after treatment with DPQ. Results from two determinations in one experiment are shown. Other details as in Figure 1.
Figure 3
Figure 3
Effect of PARP-1 inhibitors on DNA end joining in Ku deficient cells using an in vivo plasmid assay. (A) Map of pEGFP-Pem1-Ad2 plasmid used. Note the HindIII and I-SceI restriction sites upstream and downstream the Ad2 exon that are used to linearize the plasmid and generate (after removal of Ad2) the ends shown in the lower part of the figure. Upon successful intracellular plasmid circularization EGFP expression is restored and quantitated by flow cytometry. (B) Two million cells were transfected with 200 ng of HindIII, or I-SceI linearized pEGFP-Pem1-Ad2 together with 200 ng supercoiled DsRed (to monitor transfection efficiency). The ratio of GFP+ to DsRed+ cells was calculated (values inserted in the individual dot plots) and used to determine the relative rejoining between samples exposed to inhibitors and samples left untreated. Results obtained with HindIII linearized plasmid in xrs6 and xrs-6/Ku80 cells, as well as with I-SceI linearized plasmid in xrs-6 cells are shown. Cells were pretreated with DMSO, 3′-AB (10 mM) and DIQ (100 μM) 1 h before electroporation and incubation with drugs continued in the 24 h allowed for repair and expression of the reporter genes. (C) Quantification of results shown in (B). Plotted is relative plasmid rejoining in xrs-6 and xrs-6/Ku80 cells treated with DMSO, 3′-AB or DIQ as calculated by dividing the GFP+/DsReD+ ratios of samples treated with inhibitors by those measured in cells treated with DMSO. Results obtained with cells transfected with HindIII, as well as I-SceI digested plasmid are shown. Included in the figure are also results obtained with the DNA–PKcs deficient mutant V3 (dot plots not shown).
Figure 4
Figure 4
Effect of PARP-1 inhibitors on plasmid DNA end joining in LIG4−/− MEFs before and after Ku knock-down. (A) Flow cytometry dot plots showing the effect of 3′-AB and DIQ on rejoining of HindIII, or I-SceI, linearized pEGFP-Pem1-Ad2 in p53−/−/LIG4−/− (200 ng plasmid), or p53−/− (wild-type) (50 ng plasmid) MEFs. Other details as in Figure 3. (B) Quantification of results shown in (A). Other details as in Figure 3. (C) Flow cytometry dot plots showing the effect of KU70 knock-down on plasmid end joining in p53−/−/LIG4−/− MEFs. The upper panel shows results of cells treated with control siRNA, while the lower panel results of cells treated with KU70 siRNA. Other details as in Figure 3. (D) Quantification of results shown in (C). Plotted is the GFP+/DsRed+ ratio for the two samples. The insert shows a western blot analyzing the level of knock-down at the time of plasmid transfection and shows Ku70 levels as well as the levels of GAPDH used as loading control. The percent knock-down achieved is also indicated. Plasmid was transfected 24 h after treatment with siRNA and flow cytometry was carried out after an additional incubation for 24 h. (E) Effect of 3′-AB on plasmid rejoining in p53−/−/LIG4−/− MEFs treated with siRNA targeting KU70, as well as a control siRNA. Other details as in Figure 3.
Figure 5
Figure 5
Effect of H2O2 treatment on plasmid DNA end joining in p53//LIG4/ MEFs. (A) Flow cytometry dot plots showing p53−/−/LIG4−/− MEFS treated for 5 min with 5 mM H2O2 at room temperature or left untreated, immediately transfected with 200 ng HindIII linearized pEGFP-Pem1-Ad2 and incubated for 24 h in the presence of DMSO, 3′-AB (10 mM) or DIQ (100 μM). DMSO treated cells were used as controls. Other details as in Figure 3. (B) Quantitative analysis of the results shown in (A). Data shown is normalized to that obtained in the absence of PARP-1 inhibitors. Other details as in Figure 3. (C) Plot of rejoining efficiency in untreated and H2O2 treated cells. The upper panel shows the cell cycle analysis of treated and non-treated cells at the time of analysis (24 h after transfection).
Figure 6
Figure 6
EMSA shows that Ku and PARP-1 compete for DNA ends. (A) EMSA was carried out by incubating the indicated amounts of Ku and anti-Ku80 antibody in the presence of 2 ng radioactively labeled OA/OB substrate for 15 min. Subsequently PARP-1 was added at the indicated amounts for 30 min, the reaction was stopped and products were analyzed by gel electrophoresis. Two bands generated by antibody mediated supershift of Ku are termed Ku-S1 and Ku-S2 and reflect loading of the substrate ends with one or two Ku molecules, respectively. The band generated by PARP-1 is also shown. (B) Same as A, but for reactions assembled with 0.5 pmol Ku and 0.7 pmol anti-Ku antibody. Note that Ku-S2 band gains intensity and that loaded Ku can be only partly displaced under these conditions. (C) Same as in A, but with reactions incubated first with 400 ng PARP-1 followed by incubation with the indicated amounts of KU and anti-KU antibody.

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