Study of cellular responses to single-strand break

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Although single-strand breaks (SSBs) occur frequently, the cellular responses and repair of SSB are not well understood. To address this, we established mammalian cell lines expressing Neurospora crassa UV damage endonuclease (UVDE), which introduces a SSB with a 3'-OH, immediately 5' to UV-induced cycrobutane, pyrimidine dimers or 6-4 photoproducts and initiates an alternative excision repair process. Xeroderma pigmentosum group A cells expressing UVDE show UV resistance of almost the wild-type level. In these cells SSBs are produced upon UV irradiation and then efficiently repaired. The repair patch size is about 7 nucleotides and repair synthesis is decreased to 30% by aphidicoline, suggesting the involvement of a DNA polymerase δ/ε-dependent long-patch repair. Immediately after UV irradiation cellular proteins are poly(ADP-ribosyl)ated. The UV resistance of the cells is decreased in the presence of 3-aminobenzamide (3AB), an inhibitor of poly- (ADP-ribose) polymerase (PARP). Expression of UVDE in XRCC1-defective EM9, a Chinese hamster ovary cell line, greatly sensitizes the host cells to UV and addition of 3AB results in almost no further sensitization of the cells to UV. Thus, we show that XRCC1 and PARP are involved in the same pathway for the repair of SSBs.