REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance. Author summary: Nucleoside analogs have been frequently used for the treatment of virus infections and cancers. These drugs restrict the proliferation of viruses and cancers by interfering with DNA replication. The drug floxuridine (5-fluorodeoxyuridine, FUdR) is a thymidine analog and used for treating hepatic metastases of colorectal cancer. However, the effect of this drug on healthy non-cancer cells and the mechanisms underlying the cellular tolerance to FUdR remain elusive. We here determined the cellular effect of FUdR and the repair system involved the cellular tolerance to FUdR. We found that REV3, a polymerase involved in translesion DNA synthesis (TLS), promotes bypass replication on a FUdR-damaged template. Moreover, REV3 promotes DNA checkpoint activation and induces cell-cycle arrest in the early S-phase, which is required for the suppression of FUdR incorporation in the genome thereby contributing to the suppression of chromosome instability. In this study, we uncovered the previously unappreciated roles of REV3; the promotion of the cellular tolerance to FUdR by activating TLS and intra-S checkpoint. [ABSTRACT FROM AUTHOR]