Department of Toxicogenetics, Leiden University Medical Center, PO box 9600, 2300 RC Leiden, The Netherlands.
Nucleic Acids Res. 2014 Apr;42(7):4406-13. doi: 10.1093/nar/gkt1412. Epub 2014 Jan 23.
Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3(-/-) and Rev1(-/-) cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3(-/-) and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.
Rev3 是 DNA 聚合酶 ζ 的催化亚基，对于细胞毒性 DNA 光损伤的跨损伤合成至关重要，而 Rev1 蛋白在跨损伤合成中发挥非催化作用。在这里，我们揭示了哺乳动物 Rev3(-/-)和 Rev1(-/-)细胞系还表现出核苷酸切除修复 (NER) 缺陷，特别是在 S 期。这种缺陷与参与 NER 早期阶段的因素在 DNA 损伤部位的正常募集但延长持久性相关，而修复合成受到影响。值得注意的是，NER 缺陷仅在辐照后 2 小时变得明显，表明 Rev3 仅间接地影响修复合成，而不是在 NER 中发挥酶作用。我们提供的证据表明，NER 缺陷是由于 Replication protein A (Rpa) 可用于 NER 的稀缺性引起的，这是由于其在停滞的复制叉处被隔离。使用羟基脲诱导复制应激也会阻止 Rpa 在光损伤部位的积累，这在 Rev3(-/-)和野生型细胞中都是如此。这些数据支持这样一种模型，即有限的 Rpa 池协调复制应激和 NER，导致当复制应激超过阈值时，紫外线的细胞毒性增加。
