3170 Nucleic Acids Research, 2011, Vol. 39, No. 8 the repair capacity of CHO cells we treated EM9-XH and EM9-V cells with increasing doses of DMS and left them to repair for 15 min (Figure 3). We found that there is a dose-dependent increase of SSBs, suggesting that the amount of damage to the DNA, at the doses of DMS used in this study, is not saturating the engaged repair pathways. MPG is required for BER initiation after DMS-induced lesions in cells In order to ascertain that the induced SSBs observed after DMS treatment represent damages processed by BER and not direct SSBs, we siRNA depleted the glycosylase MPG in A549 cells and determined the efﬁciency of the MPG knockdown (MPGKD) by western blotting (Figure 4A). Subsequently, we followed the SSB formation in MPGKD cells after DMS treatment and found that MPGKD cells exhibited SSBs at background level through- out the time of repair (Figure 4B), in contrast to cells with functional MPG. A similar differential effect was seen when both DMS treatment and the following repair were carried out in the presence of a PARP inhibitor, indicating that the majority of SSBs produced after DMS treatment arise from MPG-dependent removal of methylated bases. BER kinetics is unaffected in PARP1 knockdown cells We found that PARP inhibition efﬁciently disrupts BER (Figures 2 and 4). This could be due to an essential role of PARP1 in the latter steps of BER ligation (20) e.g. because PARP inhibitors will block the release of PARP1 from the 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. SSB intermediate it is supposed to protect. To test this we siRNA depleted PARP1 in A549 cells and determined the efﬁciency of the PARP1 knockdown (PARP1KD) by western blotting (Figure 5A). We followed the repair of SSBs in PARP1KD cells after DMS treatment and found that PARP1KD cells exhibited a slightly lower amount of SSBs after DMS treatment than wild-type A549 cells (Figure 5B), indicating that PARP1 is in fact not necessary for ligation. Interestingly, the overall SSB levels in PARP1KD cells were slightly closer to background compared to wild-type A549 cells after DMS treatment. Next, we followed the SSB levels in A549 and PARP1KD cells exposed to DMS and incubated in the presence of the PARP inhibitor. We found an accumulation of SSBs in the wild-type cells but not in the PARP1KD cells (Figure 5C). Also, the overall background levels were increased following addition of a PARP inhibitor in DMS treated cells, which in speculation could reﬂect the role of PARP2 in BER or residual PARP1 protein in these cells. BER continues for at least 2 h after the induction of alkylation lesions Finally, we wanted to investigate the kinetics of BER and analyze remaining repair when a large portion of the alkylated DNA has already been repaired. To this end we treated EM9-XH cells with DMS (2 mM) for 15 min on ice as described, washed the cells thoroughly, and investigated the level of SSBs during repair. We found that the level of SSBs increases signiﬁcantly when adding a PARP inhibitor 2 h after the DMS treatment is terminated (Figure 6A). Control experiments with un- treated cells showed that the amount of SSBs visualized after addition of the PARP inhibitor does not reﬂect endogenous SSBs (Figure 6B). We also followed the repair of SSBs in MPGKD cells after DMS treatment (0.5 mM) and found that MPGKD cells exhibited background levels of SSBs which remain low throughout the time of repair (Figure 6C), in contrast to cells with functional MPG. Addition of a PARP inhibitor 2 h after removal of DMS increased SSB levels more prominently in wild-type as compared to MPGKD cells (Figure 6C), demonstrating that the formation of late SSBs is dependent on active BER. Untreated control cells did not display any change in SSB levels, even after the addition of PARP inhibitor 2 h mock treatment is terminated (Figure 6D). DISCUSSION Here, we conﬁrm the importance of both XRCC1 and PARP in the repair induced by oxidative damage to the DNA (Figure 1A, D and E). XRCC1 is necessary for repair after induction of oxidative damage to the DNA, most likely by recruiting and stabilizing Ligase IIIa (19). PARP inhibition also has a decelerating effect on the rate of ligation, but the two proteins clearly have distinct roles in this repair, as PARP inhibition further delay SSB repair in XRCC1 defective cells. We ﬁnd that more than half of the H2O2-induced SSBs are