Nishizawa Eri, Ohkubo Hiromori, Kawasumi Ryotaro, Tsuda Masataka, Hirota Kouji
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.
Division of Genome Safety Science, National Institute of Health Science, 3-25-26 Tonomachi, Kawasaki-shi, Kanagawa, 210-9501, Japan.
DNA Repair (Amst). 2025 Aug 14;153:103885. doi: 10.1016/j.dnarep.2025.103885.
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 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, and POLE1 cells exhibited similar sensitivity to ddC, AZT, and alovudine, whilst POLD1/POLE1 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.
链终止核苷类似物（CTNAs）在复制过程中被复制性聚合酶δ（Polδ）和ε（Polε）掺入基因组，然后通过阻止后续聚合反应来抑制DNA合成。Polε的校对核酸外切酶活性可去除掺入的CTNAs，从而有助于细胞对这些药物产生耐受性。然而，Polδ的校对核酸外切酶活性的作用尚未阐明，Polδ与Polε之间的关系也未得到充分理解。我们在此表明，Polδ的核酸外切酶活性有助于细胞对CTNAs产生耐受性，Polδ和Polε核酸外切酶的作用因CTNAs的种类而异。我们测试了POLD1细胞对CTNA阿糖胞苷（Ara-C）的敏感性，发现核酸外切酶缺陷型Polδ的表达使细胞对Ara-C敏感。此外，在Polε核酸外切酶功能正常和缺陷的细胞中，核酸外切酶缺陷型Polδ均使Ara-C处理后的细胞活力降低至相同程度，这表明这两种聚合酶独立地有助于细胞对Ara-C产生耐受性。相比之下，野生型、POLD1和POLE1细胞对双脱氧胞苷（ddC）、齐多夫定（AZT）和阿洛苷表现出相似的敏感性，而POLD1/POLE细胞与这些细胞相比则明显更敏感，这表明Polδ和Polε相互补偿。最后，我们发现复制性聚合酶的核酸外切酶活性无法从新生DNA末端去除阿昔洛韦（ACV）。综上所述，我们的研究结果表明，CTNAs对复制叉有不同的影响，复制性聚合酶核酸外切酶活性的需求因CTNAs种类而异。
