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Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells
bioRxiv - Molecular Biology Pub Date : 2020-10-12 , DOI: 10.1101/2020.10.12.336107
Yann Benureau , Caroline Pouvelle , Eliana Moreira Tavares , Pauline Dupaigne , Emmanuelle Despras , Eric Le Cam , Patricia Kannouche

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

中文翻译:

复制中间结构揭示了人类细胞中紫外线老化的复制叉处的DNA损伤耐受途径的时间顺序

S期的DNA损伤威胁基因组稳定性。DNA损伤耐受(DDT)途径克服了这些障碍,并可以通过使用特殊的转移(TLS)DNA聚合酶或通过与重组相关的过程来完成DNA合成。但是,这些机制之间如何相互配合以及与批量复制之间如何协调仍然难以捉摸。为了解决这些问题,我们使用透射电子显微镜监测了响应紫外线照射的复制中间体结构的变化。我们显示,TLS聚合酶η能够准确绕过主要的紫外线损伤,并在皮肤癌多发性干性色素变种(XPV)综合征中发生突变,在复制叉处起作用,以解决解偶联并防止复制后间隙积累。当这种即时机制无法运行时,引发作为补偿机制而发生,因此在XPV细胞中占主导地位。有趣的是,我们的数据在复制叉处支持RAD51的重组独立功能,以维持重新启动。最后,我们为缺口修复中重组后的复制承诺和体内重组中间体的开拓性观察提供了证据。总而言之,我们提出了人类细胞中紫外线损伤耐受的时间顺序,强调了poln在塑造这种反应和确保DNA合成的连续性中的关键作用。我们为间隙修复中重组后的复制承诺以及体内重组中间体的开拓性观察提供了证据。总而言之,我们提出了人类细胞中紫外线损伤耐受的时间顺序,强调了poln在塑造这种反应和确保DNA合成的连续性中的关键作用。我们为间隙修复中重组后的复制承诺以及体内重组中间体的开拓性观察提供了证据。总而言之,我们提出了人类细胞中紫外线损伤耐受的时间顺序,强调了poln在塑造这种反应和确保DNA合成的连续性中的关键作用。
更新日期:2020-10-13
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