Analysis of regulatory mechanisms of translesion DNA synthesis in human cells

我们已经确定了人类DNA聚合酶eta（pol eta）作为着色性干皮病变异体（XPV）的责任基因产物，XPV是一种紫外线敏感的遗传性疾病，伴有癌症倾向。我们已经表明，聚合酶催化相对准确translesion DNA合成过去的环丁烷嘧啶二聚体，最常见的病变紫外线照射诱导的DNA。然而，Pol eta从来都不是精确的DNA聚合酶，如果它复制了未受损的DNA模板，它实际上会诱导突变。这些先前的观察有力地表明，细胞必须有调节机制来控制细胞。为了解决这些问题，研究了pol eta和相关蛋白之间的蛋白质-蛋白质相互作用，并将Rev 1和Rad 6/Rad 18复合物鉴定为与pol eta相互作用的蛋白质。Rev 1重新定位细胞中的UV诱导的损伤以及pol eta。重要的是，Rev 1在受损DNA位点的积累在很大程度上依赖于聚合酶。已知Rev 1除了与pol eta相互作用外，还与其他跨损伤DNA聚合酶相互作用。因此，Rev 1可能在跨损伤聚合酶之间的聚合酶转换中起重要作用。pol eta和Rad 6/Rad 18以及Rev 1的相互作用通过UV照射或某些药物诱导复制停滞而在染色质DNA上增强。Rad 6/Rad 18负责PCNA的单泛素化，PCNA是DNA复制聚合酶的滑动夹。这种修饰被认为是聚合酶在复制性聚合酶和跨损伤聚合酶之间切换的关键事件。这些结果为了解跨损伤合成的调控机制提供了重要的分子证据。

We have identified human DNA polymerase eta (pol eta) as the responsible gene product of xeroderma pigmentosum variant (XPV), an ultraviolet light sensitive genetic disease accompanied with cancer proneness. We have shown that pol eta catalyzes relatively accurate translesion DNA synthesis past cyclobutane pyrimidine dimers, the most frequent lesions induced by UV-irradiation to DNA. Pol eta, however, is never accurate DNA polymerase and actually induces mutations if it replicated undamaged DNA templates. These previous observations strongly indicate that cells must have regulatory mechanisms to control pol eta. To address them, protein-protein interactions between pol eta and related proteins are examined and identified Rev1 and Rad6/Rad18 complex as interacting proteins with pol eta. Rev1 re-localizes UV-induced lesions in cells as well as pol eta. Importantly, accumulation of Rev1 to damaged DNA locus is largely dependent on pol eta. Rev1 is known to interact with other translesion DNA polymerases in addition to pol eta. So, it is likely that Rev1 plays an important role in polymerase switches between translesion polymerases. Interaction of pol eta and Rad6/Rad18, and also Rev1, is enhanced on the chromatin DNA by inducing replication arrests by UV-irradiation or some drugs. Rad6/Rad18 is responsible for mono-ubiquitylation of PCNA, the sliding clamp for DNA replication polymerases. The modification is thought to be an key event for the polymerase switch between replicative and translesion polymerases. These results gave a great molecular evidences to understand regulatory mechanisms of translesion synthesis in human cells.