The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C).
The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C).
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DOI：
10.18632/oncotarget.16508
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发表时间：
2017-05-16
期刊：
影响因子：
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通讯作者：
Hirota K
中科院分区：
文献类型：
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作者：
Tsuda M;Terada K;Ooka M;Kobayashi K;Sasanuma H;Fujisawa R;Tsurimoto T;Yamamoto J;Iwai S;Kadoda K;Akagawa R;Huang SN;Pommier Y;Sale JE;Takeda S;Hirota K
阿糖胞苷、5-氟尿嘧啶（5-FU）和曲氟尿苷（FTD）等化学修饰的核苷类似物经常通过复制型DNA聚合酶掺入DNA中。然而，目前还不清楚这种掺入如何杀死循环细胞。有两种可能：核苷类似物三磷酸抑制复制型DNA聚合酶，和/或错误掺入基因组DNA中的核苷酸类似物干扰下一轮DNA合成，因为复制型DNA聚合酶将它们识别为模板DNA损伤，从而阻止合成。为了解决第一种可能性，我们选择性地破坏DNA聚合酶ε（Polε）的校正核酸外切酶活性，DNA聚合酶ε是禽类DT 40和人类TK 6细胞系中的前导链复制聚合酶。为了解决第二个问题，我们破坏了RAD 18，这是一种参与translesion DNA合成的基因，这是一种缓解停滞复制的机制。值得注意的是，POLE 1 exo −/−细胞对阿糖胞苷超敏，而RAD 18 −/−细胞对FTD超敏。Ara-C脉冲后的gH 2AX病灶形成是立即的，并且没有进展到下一轮复制，而5-FU和FTD脉冲后的gH 2AX病灶形成延迟到下一轮复制。生物化学研究表明，人类校正缺陷型Polε-exo−全酶结合了Ara-CTP，但随后从该碱基延伸的效率比从完整核苷酸延伸的效率低几倍。总之，我们的结果表明，Ara-C通过阻断新生DNA链的延伸起作用，并被Polε的校正活性抵消，而5-FU和FTD被有效掺入，但在随后的S期作为复制叉阻断剂，这被跨损伤合成抵消。
Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo−/− cells, but not RAD18−/− cells, were hypersensitive to Ara-C, while RAD18−/− cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo− holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.