3168 Nucleic Acids Research, 2011, Vol. 39, No. 8 absence or presence of a PARP inhibitor. As expected, we found that both XRCC1 deﬁciency as well as co-treatment with PARP inhibitor sensitizes cells to hydrogen peroxide (Figure 1A). Interestingly, we report that XRCC1 defect- ive EM9-V cells are hypersensitive to PARP inhibitors alone (Figure 1B). In order to investigate whether this hypersensitivity is due to the functions of PARP and XRCC1 in SSB repair, we measured the background level of SSBs in these cells. To allow for a direct and quantitative measurement of the number of SSBs we employed the ADU technique (13,14,17), and we found no correlation with the background levels of SSBs and the presence of XRCC1 or the addition of a PARP inhibitor (Figures 1C and 6B). When monitoring SSB repair by measuring the level of SSBs in cells over time after H2O2-induced damage, we found that PARP inhibition reduce the rate of SSB rejoin- ing (Figure 1D), which support previous results obtained with the comet assay (15). Also, we conﬁrmed that the level of SSBs is considerably enhanced in the cell line lacking XRCC1, indicating impaired ligation of breaks (Figure 1E). PARP inhibition in these cells had an add- itional decelerating effect on the rate of ligation, further indicating distinct roles of the two proteins in SSB repair. 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 mM), 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 mM). 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 mM 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.