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Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage

机译：人DNA聚合酶δ的时空募集到紫外线损伤部位

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摘要

Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol δ subunits to sites of DNA damage produced by UV irradiation through 5 μm polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol δ lacking the p12 subunit. ChIP analysis of Pol δ after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol δ3 is the predominant form of Pol δ at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol δ was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol δ at the DNA damage site is the Pol δ trimer lacking p12 regardless of the cell cycle phase.
机译：人类DNA聚合酶δ（Polδ）除了在DNA复制中起重要作用外，还参与各种DNA损伤反应。 Polδ4全酶由四个亚基p125，p50，p68和p12组成。已经确定，p12亚基响应UV对DNA的损伤而迅速降解，导致体内Polδ4向Polδ3的转化，这是缺少p12亚基的三聚体形式。我们提供了通过免疫荧光显微镜和激光扫描细胞术（LSC）通过5μm聚碳酸酯滤光片紫外线照射产生的单个Polδ亚基到DNA损伤部位的时间依赖性募集的首次分析。定量分析表明，三个大亚基的募集到2 h几乎完成，并且在暴露于紫外线后4 h内没有明显变化。但是，即使在4 h时，p12的募集仍不完全，大约70％的Polδ缺乏p12亚基。全局紫外线照射后，Polδ的ChIP分析进一步证明仅存在p125，p50和p68。因此，由于p12降解，Polδ3是UV损伤部位Polδ的主要形式。使用LSC，我们进一步证实了Polδ在细胞周期的所有阶段都被招募到CPD损伤部位。总体而言，我们的结果表明，DNA损伤位点的Polδ是缺少p12的Polδ三聚体，而与细胞周期阶段无关。

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