Analysis of regulatory mechanisms of translesion DNA synthesis in human cells

我们已经确定人类DNA聚合酶eta （pol eta）是色素干皮病变异（XPV）的负责基因产物，XPV是一种伴随癌症易感性的紫外光敏感遗传疾病。我们已经证明，pol - eta催化相对准确的翻译DNA合成过去环丁烷嘧啶二聚体，最常见的损伤由紫外线照射到DNA。然而，Pol - eta从来不是精确的DNA聚合酶，如果它复制未受损的DNA模板，实际上会引起突变。这些先前的观察结果有力地表明细胞必须有调控机制来控制pol - eta。为了解决这些问题，我们研究了pol eta和相关蛋白之间的蛋白-蛋白相互作用，并确定了Rev1和Rad6/Rad18复合物是与pol eta相互作用的蛋白。Rev1重新定位紫外线诱导的细胞病变以及pol - eta。重要的是，Rev1在受损DNA位点的积累很大程度上依赖于pol - eta。Rev1已知与除pol - eta外的其他翻译DNA聚合酶相互作用。因此，Rev1可能在翻译聚合酶之间的聚合酶转换中起重要作用。pol - eta和Rad6/Rad18以及Rev1在染色质DNA上的相互作用通过紫外线照射或某些药物诱导复制阻滞而增强。Rad6/Rad18负责PCNA的单泛素化，PCNA是DNA复制聚合酶的滑动钳。这种修饰被认为是聚合酶在复制聚合酶和翻译聚合酶之间转换的关键事件。这些结果为理解人细胞翻译合成的调控机制提供了重要的分子依据。

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.