3172 Nucleic Acids Research, 2011, Vol. 39, No. 8 Figure 5. BER kinetics is unaffected in PARP1 knockdown cells. (A) Western blot, probed with antibodies against PARP1 and loading control b-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. PARP1 not having a direct role in BER. In the literature, PARP1 is denoted as a BER protein, which is likely related to previous observations that cell extracts from PARP1 / cells are unable to complete the DNA synthesis step of BER in vitro (20). However, the presence of PARP1 is not required for effective BER in an in vitro biochemical assay, and may even decrease the biochemical kinetics of BER (24). So far, it has not been possible to assign an exact function of PARP1 in BER (24), but our data support a model where BER does not require PARP1. This is also in line with PARP1 protein being poorly conserved while many other BER factors are well conserved through evolution. The model proposes that the subset of BER SSB intermediates that become uncoupled somewhere during the repair pathway, are bound by PARP1 when it is present in the cell (Figure 7). When PARP is inhibited it is thereby trapped on these SSB intermediates, thus blocking their ligation and causing a potentially toxic retention of SSBs in the DNA. This would explain the increase of DMS- induced SSBs observed following PARP inhibition. Such trapping of PARP to SSB intermediates may explain the high toxicity of PARP inhibitors in BRCA2 defective cells, in contrast to the very modest toxicity of co-depletion of BRCA2 and PARP1 (25). There are two competing models for BER. One model separate the BER incision and the subsequent SSB repair, meaning that the DNA lesion would be identiﬁed twice; ﬁrst by a glycosylase and then by PARP1 for SSB repair. The other model suggests that the DNA lesion is only recognized by a glycosylase and all following steps are coordinated (Figure 7) (26–28). Since we ﬁnd no accumu- lation of the SSB intermediate in PARP1 siRNA depleted cells, we suggest that the DNA lesion is only identiﬁed by glycosylases/endonucleases and that the following steps are coordinated. BER of DMS induced damages appears to be a fast and efﬁcient repair pathway as the majority of the SSB inter- mediates are incised and ligated within 30 min after exposure to the DNA damaging agent (Figures 2B, 4B and 6A). However, we ﬁnd that a large number of repair events (as measured by SSB formation after addition of a