SMUG1 but not UNG DNA glycosylase contributes to the cellular response to recovery from 5-fluorouracil induced replication stress.

5-Fluorouracil (5-FU) is a widely utilized cancer chemotherapeutic that causes DNA damage via two mechanisms. Its active metabolite inhibits thymidylate synthase, which deprives cells of TTP and causes the introduction of uracil in DNA. Also, 5-FU is directly incorporated into DNA. Both uracil and 5-FU in DNA are recognized by uracil-DNA glycosylases (UDGs), which initiate base excision repair. UNG and SMUG1 are the two human UDGs most likely to combat the genomic incorporation of uracil and 5-FU during replication. In this study, we examined the roles of UNG and SMUG1 in the initial cellular response to 5-FU and compared continuous exposure to a 24h exposure followed by incubation in drug-free media, which mimics what occurs clinically. Loss of UNG did not alter cellular sensitivity to 5-FU in two human cell lines, despite its predominant biochemical activity for uracil and 5-FU in DNA. Loss of SMUG1 corresponded with >2-fold increase in sensitivity to 5-FU, but only with a 24h treatment followed by recovery. There was no difference between SMUG1 proficient and depleted cells following continuous exposure. We observed that 5-FU treatment induced an enhanced S-phase arrest and CHK1 activation plus an increase in the formation of strand breaks and alkali-labile sites in all sublines. However, SMUG1-depleted cells showed a prolonged S-phase arrest, a transient increase in DNA double-strand breaks following 5-FU treatment and an altered phosphorylation of CHK1 following removal of drug. Collectively, the results suggest that SMUG1 has a role in the resumption of replication following 5-FU treatment.