Proofreading exonuclease activities of Polδ and Polε differentially contribute to the removal of chain-terminating nucleoside analogs

Chain-terminating nucleoside analogs (CTNAs) are incorporated into genome during replication by replicative polymerase delta (Polδ) and epsilon (Polε), then inhibit DNA synthesis by preventing subsequent polymerization. The proofreading exonuclease activity of Polε removes the incorporated CTNAs, thereby contributing to cellular tolerance to these drugs. However, the contribution of Polδ's proofreading exonuclease activity has not been clarified, nor has the relationship between Polδ and Polε been well understood. We here show that Polδ's exonuclease activity contributes to the cellular tolerance to CTNAs, with the role of Polδ and Polε exonucleases differing depending on the kinds of CTNAs. We tested the sensitivity of POLD1^exo-/+ cells to a CTNA, Ara-C, and found that expression of the exonuclease deficient Polδ sensitizes cells to Ara-C. Furthermore, the exonuclease deficient Polδ reduced cell viability upon Ara-C to the same extent in both Polε exonuclease-proficient and -deficient cells, indicating that these two polymerases independently contribute to cellular tolerance to Ara-C. In contrast, wild-type, POLD1^exo-/+, and POLE1^exo-/- cells exhibited similar sensitivity to ddC, AZT, and alovudine, whilst POLD1^exo-/+/POLE1^exo-/- cells were considerably more sensitive compared with these cells, indicating that Polδ and Polε compensate for each other. Finally, we found that exonuclease activities of replicative polymerases cannot remove ACV from the end of nascent DNA. Taken together, our findings show that CTNAs have a differential impact on the replication fork, and the requirement of the exonuclease activities of replicative polymerases varies depending on the kinds of CTNAs.