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先导链 DNA 聚合酶 epsilon 的校对外切酶可防止断裂模板链处的复制叉崩溃。

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

机构信息

Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan.

出版信息

Nucleic Acids Res. 2023 Dec 11;51(22):12288-12302. doi: 10.1093/nar/gkad999.

DOI:10.1093/nar/gkad999
PMID:37944988
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10711444/
Abstract

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

摘要

聚合酶 epsilon (Polϵ) 在单链断裂 (SSB) 处进行领头链 DNA 复制会导致单端双链断裂 (seDSB)，这些断裂通过同源定向修复 (HDR) 进行修复，并受到叉形反转 (FR) 的抑制。尽管先前的研究确定了许多羟基脲诱导 FR 所需的分子，但 seDSB 处的 FR 仍知之甚少。在这里，我们鉴定了专门介导 seDSB 处 FR 的分子。由于 seDSB/FR 相关分子需要多聚 ADP 核糖聚合酶 1 (PARP1)，我们假设 seDSB/FR 相关分子将增加对喜树碱 (CPT) 的耐受性，但不会增加对 PARP 抑制剂奥拉帕利的耐受性，尽管这两种抗癌药物都会产生 seDSB。事实上，我们发现 Polϵ 核酸外切酶和 Polϵ 辅助因子 CTF18 增加了对 CPT 的耐受性，但对奥拉帕利没有影响。为了探索 Polϵ 核酸外切酶、CTF18 和 PARP1 之间潜在的功能相互作用，我们构建了核酸外切酶缺陷型 POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, 和 CTF18-/-/PARP1-/- 细胞。上位性分析表明 Polϵ 核酸外切酶和 CTF18 是相互依赖的，并且需要 PARP1 来耐受 CPT。值得注意的是，POLE1exo-/- 和 HDR 缺陷型 BRCA1-/- 细胞表现出相似的 CPT 敏感性。此外，将 POLE1exo-/- 与 BRCA1-/- 突变相结合会协同增加 CPT 敏感性。总之，新鉴定的 PARP1-CTF18-Polϵ 核酸外切酶轴和 HDR 独立地作用以防止 seDSB 处的叉形崩溃。奥拉帕利抑制该轴，这解释了奥拉帕利对 HDR 缺陷型细胞的显著细胞毒性作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/820010f4fea4/gkad999fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/4b7e5ad5efc9/gkad999figgra1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/e434246b3123/gkad999fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/856357579d9d/gkad999fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/96c3b57be7a3/gkad999fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/d7431a635f74/gkad999fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/18ef13e788b7/gkad999fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/90ce9c25b0fa/gkad999fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/820010f4fea4/gkad999fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/4b7e5ad5efc9/gkad999figgra1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/e434246b3123/gkad999fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/856357579d9d/gkad999fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/96c3b57be7a3/gkad999fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/d7431a635f74/gkad999fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/18ef13e788b7/gkad999fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/90ce9c25b0fa/gkad999fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fade/10711444/820010f4fea4/gkad999fig7.jpg

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