3166–3175 Nucleic Acids Research, 2011, Vol. 39, No. 8 doi:10.1093/nar/gkq1241 Published online 22 December 2010 Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate Cecilia E. Stro¨ m1, Fredrik Johansson 1 , Mathias Uhle´ n2, Cristina Al-Khalili Szigyarto2, Klaus Erixon1 and Thomas Helleday1,3,* 1Department of Genetics, Microbiology and Toxicology, Stockholm University, 2Department of Proteomics, School of Biotechnology, Royal Institute of Technology/AlbaNova, S-106 91 Stockholm, Sweden and 3Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford, OX3 7DQ, UK Received June 19, 2010; Revised November 11, 2010; Accepted November 12, 2010 ABSTRACT Base excision repair (BER) represents the most im- portant repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid ac- cumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point. INTRODUCTION Base damages, such as methylations, oxidations, depurinations and single-strand breaks (SSBs) are commonly formed by endogenous cellular metabolism (1). The recognition of these damages is imperative for efﬁcient repair and is achieved by a set of specialized glycosylases. Poly(ADP-ribose)polymerase 1 (PARP1) is an evolutionary poorly conserved nick-sensing enzyme with the catalytic ability to produce long polymers of ADP-ribose on itself and other proteins (2). This polymer- ization is stimulated by the binding of PARP1 to a nick in the DNA and results in the rapid relocation of repair proteins such as XRCC1, to the site of the lesion (3). PARP1 is known to interact with proteins in both short- and long-patch repair pathways downstream of damage recognition (4–7). Precisely how PARP1 functions in repair and signalling is not yet determined as its main activity at the DNA SSB appears to be autoribosylation, which is ﬁnalised when the negatively charged ADP-ribose polymers cause PARP1 to dissociate from the DNA. Inhibition of PARP1 has been shown to impair DNA SSB repair, as the inhibition of its polymeric activity traps the enzyme at the SSB and physically blocks further repair (8). However, PARP1 does not appear to be required for SSB repair as cells with PARP1 knocked down still display efﬁcient SSB repair activity (8). The binding of PARP1 to SSBs may act as protection from excessive DNA damage by sequestering the potentially toxic intermediates until they can be repaired. Although PARP1 is often annotated to be a base excision repair (BER) protein it is unclear exactly how and if PARP1 is involved in the repair of DNA lesions such as methylated or oxidizes bases. Here, we hypothesize that PARP1 has no active role in BER, since it is a poorly conserved protein through evolution, in contrast to many other BER factors. Furthermore, the aim of this study is to assay SSB formation to enable us to investigate the impact of PARP1 in the repair of methylated DNA and to investigate how PARP inhibition and knockdown can affect this process. *To whom correspondence should be addressed. Tel: +46 8 16 29 14; Fax: +46 8 16 43 15; Email: helleday@gmt.su.se The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.