Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Abstract

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

Figure 1.

PARP1 and XRCC1 have distinct roles in efficient SSB repair. The PARP1 and XRCC1 proteins are involved in DNA repair in response to hydrogen peroxide. (A) Survival, following a 15 min exposure to hydrogen peroxide on ice, of XRCC1 deficient EM9 cells expressing wild-type XRCC1 (EM9-XH) or empty vector (EM9-V). Cells were treated in the presence or absence of PARP inhibitor (1,8-napthalimide, 50 µM), which was left in the growth medium for 24 h after treatment. Survival is plotted as percentage of living mock treated cells without PARP inhibition. The means and standard errors of three experiments are shown. (B) XRCC1 defective EM9-V cells are hypersensitive to PARP inhibitors alone. Survival of EM9-XH and EM9-V cells, following a 24 h treatment with PARP inhibitor under normal growth conditions. The means and standard errors of three experiments are shown. (C) Background levels of SSBs in EM9-V or EM9-XH cell lines, measured after various time points of incubation with or without PARP inhibitor (50 µM). The means and standard errors of three experiments are shown. SSB repair rates in the cell lines EM9-XH (D) and EM9-V (E) after a 15 min treatment with 200 µM hydrogen peroxide. Dotted lines indicate the levels of SSBs in mock treated cells for each cell line. Cells were treated and left to repair in the presence or absence of PARP inhibitor. The means and standard errors of three experiments are shown.

Figure 2.

Distinct roles of XRCC1 and PARP inhibition in BER. (A) To study SSB formation during BER, cells were seeded 24 h prior to labelling the DNA with ³H-TdR for an additional 24 h. Exposure to DMS was performed on ice to reduce repair during treatment, where after DNA repair was induced by raising the temperature to 37°C for various time intervals. The amount of strand breaks was measured by the alkaline DNA unwinding technique (12). BER incision and formation of a SSB is independent of XRCC1 in cells. Levels of SSBs in the EM9-V and EM9-XH cell lines after a 15 min treatment with 0.5 mM DMS at indicated time points of repair. Cells were treated and left to repair in the (B) absence or (C) presence of PARP inhibitor. The means and standard errors of three experiments are shown. (D) Survival, following a 15 min exposure to DMS on ice, of XRCC1 deficient EM9-V cells expressing empty vector (EM9-V) or wild-type XRCC1 (EM9-XH). Cells were treated in the presence or absence of PARP inhibitor, which was left in the growth medium for 24 h after treatment. Survival is plotted as percentage of living mock treated cells without PARP inhibition. The means and standard errors of three experiments are shown.

Figure 3.

DMS dose-curve of induced SSBs. SSBs were measured in EM9-V and EM9-XH cells treated with increasing doses of DMS and left to repair for 15 min in fresh DMEM. The means and standard errors of two experiments are shown.

Figure 4.

The glycosylase MPG is required for the formation of the majority of SSBs produced during the repair of DMS-induced damages. (A) Western blot, probed with antibodies against MPG and loading control β-tubulin, showing the siRNA knockdown of MPG in human A549 cells. (B) Levels of SSBs in wild-type A549 cells and cells depleted of MPG, after a 15 min treatment with 0.5 mM DMS at indicated time points of repair. Cells were treated and left to repair in the absence or presence of PARP inhibitor. The means and standard errors of three experiments are shown.

Figure 5.

BER kinetics is unaffected in PARP1 knockdown cells. (A) Western blot, probed with antibodies against PARP1 and loading control β-tubulin, showing the knockdown of PARP1 using siRNA in human A549 cells. Level of SSBs in A549 cells with and without siRNA knockdown of PARP1, after a 15 min treatment with 0.5 mM DMS at indicated time points of repair. Cells were treated and left to repair in the (B) absence or (C) presence of PARP inhibitor. The means and standard errors of three experiments are shown.

Figure 6.

A substantial amount of damages are still being repaired 2 h after exposure to DMS. Level of SSBs in EM9-XH cells treated with (A) DMS (2 mM) or (B) mock treated and left to repair. PARP inhibitor was added (open symbols, time point indicated by arrow) to slow down the ligation step, 2 h after treatment was terminated. The means and standard errors of three experiments are shown. The ongoing SSB formation 2 h after terminated treatment, represents MPG initiated BER events. Level of SSBs in A549 cells with or without siRNA depleted MPG and treated with (A) DMS (0.5 mM) or (B) mock treated and left to repair. PARP inhibitor was added (open symbols, time point indicated by arrow) to slow down the ligation step, 2 h after treatment was terminated. The means and standard errors of three experiments are shown.

Figure 7.

Model for BER and the influence of PARP. The MPG glycosylase is recognizing N-methylated purines, likely through scanning the DNA for base lesions. Once a lesion is recognized, MPG excises the damaged base and the APE1 endonuclease cleaves the newly formed AP-site into a SSB intermediate. If this intermediate is uncoupled from the repair process before repair is finished, PARP1 may recognize and bind to the SSB intermediate, which overall might slow down the BER process. In the event that PARP1 is inhibited it will be trapped on the SSB and prevent ligation. However, PARP1 is not required for accurate repair if the repair pathway remains intact. In the event of the BER complex being unable to accurately ligate, long patch repair is thought to take over. Our model does not exclude an active role for PARP1 in BER by influencing DNA damage-induced chromatin remodelling.