Distinct roles of XRCC1 and PARP in the repair of alkylated DNA lesions DMS is used in these experiments since it is 25–30 times more potent than methyl methanesulfonate (MMS) but will produce similar proportions of methylated DNA products (18). DMS will thus remain active on ice, as opposed to MMS. By exposing cells to DMS on ice, we ensure that a minimal background repair is operating during treatment and we can investigate whether XRCC1 and PARP have additional roles in BER, as opposed to their known functions in SSB repair (Figure 2A). We found that SSBs rapidly accumulate in EM9-V cells after DMS treatment (Figure 2B) suggesting that the de- tection and excision of methylated bases is independent of XRCC1. The ADU technique might not fully distinguish between AP-sites and SSBs formed during repair, so we cannot specify if the same is true for BER incisions. Nucleic Acids Research, 2011, Vol. 39, No. 8 3169 Furthermore, the accumulation of SSBs highlights the im- portance of the XRCC1–LigIIIa complex in the latter ligation step of both BER and SSB repair (19). In the SSB assay performed here we also observed a dramatic increase in the level of SSBs following DMS treatment in PARP inhibited cells (Figure 2C). Additionally, when impeding both proteins simultaneously by inhibiting PARP in XRCC1 deﬁcient cells we detected higher levels of SSBs than if either protein is incapacitated alone. The lack of XRCC1 sensitized cells to alkylating damage on the DNA, in a similar fashion as PARP inhibition (Figure 2D), suggesting that these proteins play a role in the repair of DMS lesions. The decreased survival in EM9-V cells treated with PARP inhibitor is most likely due to the hypersensitivity mentioned earlier (Figure 1B). The low levels of SSBs detected in wild-type cells after treatment with DMS indicate a very fast and efﬁcient repair of alkylating damages to the DNA. To determine 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.