Nucleic Acids Research, 2011, Vol. 39, No. 8 3171 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 b-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. repaired within 5 min after damage induction in the reconstituted wild-type cells (Figure 1D), showing that this repair is a fast process. It should be noted that this repair includes the BER of oxidized bases as well as SSB repair, as H2O2 produces a variety of lesions to the DNA (21). However, we see a characteristic rapid repair curve where the majority of SSBs are present in the cells directly after treatment and not after enzymatically-induced inci- sions, as can be seen after treatment with DMS. Thus, we suggest that the majority of the breaks originate from direct SSBs. We also ﬁnd that XRCC1 defective cells are hypersen- sitive to PARP inhibitors (Figure 1B) in spite of the fact that there was no statistically signiﬁcant increase of the background levels of SSBs in these cells (Figure 1C), suggesting that the increased sensitivity of XRCC1 defect- ive cells to PARP inhibitors is explained by functions of PARP1 and XRCC1 in processes apart from SSB repair. For instance, blocked replication forks activates PARP1 for efﬁcient restart to occur (22) and both PARP1 and XRCC1 have been shown to have speciﬁc roles at replication forks in S phase cells (8,22,23). In speculation, there is a possibility that inhibition of PARP would generate a toxic lesion at replication forks that requires XRCC1 for repair, or vice versa. Also, it should be noted that we have not observed any indications of this hypersensitivity affecting the cells viability during the short time of treatment and repair that are employed in this study. After conﬁrming the importance of functional XRCC1 and PARP in the repair of SSBs, we move on to compare and evaluate the role of PARP in the repair of alkylated DNA damages. By utilizing XRCC1 deﬁcient cells and a PARP inhibitor, we efﬁciently block the ligation of DNA breaks and thus we can monitor early repair events. After DMS treatment we ﬁnd a high level of SSBs in XRCC1 deﬁcient cells as well as in PARP in- hibited cells, suggesting that neither XRCC1 nor PARP are necessary for the detection or excision of alkylated DNA damages repaired by BER (Figure 2B and C). Additionally, we see even higher levels of SSBs when impeding both proteins simultaneously by inhibiting PARP in XRCC1 deﬁcient cells, leading to the conclusion that the inhibition of PARP does not affect the repair of DMS induced damages in the same manner as XRCC1 deﬁciency does. We conﬁrm that the subsequent SSBs induced by DMS predominantly originate from base alkylations, which are detected by N-methylpurine-DNA glycosylase (MPG). The level of SSBs is considerably reduced when MPG is knocked down by siRNA depletion (Figure 4B). Importantly, as no or very few SSBs are formed after DMS treatment in MPG siRNA depleted cells, our data demonstrate that MPG is the main (and possibly the only) glycosylase to recognize and excise DMS-induced base damage from DNA. The role of PARP1 is likely to recognize SSBs or other DNA ends in the DNA and to increase the repair. We ﬁnd that H2O2-induced SSB repair is retarded in cells when treated with a PARP inhibitor, as a likely consequence of reduced recruitment of XRCC1 (8). Furthermore, we ﬁnd that siRNA depletion of PARP1 did not affect the amount of DMS-induced SSBs, or if anything decreased the amount of SSBs (Figure 5B). This can be explained by