PARP1 safeguards nascent DNA from SMUG1-dependent processing of incorporated 5-hydroxymethyl-2′-deoxyuridine

DNA base damage is the most common form of lesion, occurring approximately 10,000 times per cell a day. Such damage arises either from the incorporation of damaged nucleotides or from direct modification of bases within DNA strand. 5-hydroxymethyl-2´-deoxyuridine (5-hmdU) is a relatively common base lesion generated through the incorporation of oxidized thymidine or through the deamination of 5-hydroxymethyl-2´-deoxycytidine, a product produced during epigenetic regulatory process. However, the mechanisms underlying the repair and cellular tolerance to 5-hmdU have not been fully elucidated. Here, we explored genome maintenance factors required for cellular tolerance to 5-hmdU using a mutant cell collection deficient in individual genome maintenance factors derived from chicken DT40 cell line. We found that PARP1 is the most critical factor for cellular tolerance to 5-hmdU. PARP1 −/− cells exhibited delayed replication fork progression following 5-hmdU exposure, indicating that DNA replication is acutely impaired upon 5-hmdU incorporation. Following the incorporation of 5-hmdU into nascent DNA, PARP1-deficient cells showed massive single strand breaks, which subsequently led to double strand breaks and ultimately apoptosis. Notably, these defects observed in PARP1 −/− cells were completely rescued by the loss of SMUG1, a DNA glycosylase responsible for the removal of 5-hmdU from genome. These findings indicate that the SMUG1-dependent processing of 5-hmdU in nascent DNA is highly toxic in the absence of PARP1. Collectively, our results reveal a previously unappreciated role of PARP1 in counteracting toxic effects of SMUG1-mediated 5-hmdU processing during DNA replication. (236 words)