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首页> 外文期刊>Journal of Molecular Biology >The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited.
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The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited.

机译：重新探讨了DNA聚合酶V介导的病变合成的生化要求。

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摘要

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.
机译：除复制性DNA聚合酶外，细胞还含有参与损伤耐受，诱变和免疫球蛋白多样性等过程的专门DNA聚合酶。在大肠杆菌中，由umuDC基因座编码的DNA聚合酶V（Pol V）参与跨病变合成（TLS）和诱变。遗传研究已确定，诱变既需要UmuC，又需要UmuD的蛋白水解产物（UmuD'）。另外，从基因上说，RecA蛋白和复制持续性因子β钳是诱变必不可少的辅助因子。在这里，我们已经重建了Pol V介导的三个常见复制阻滞性病变的旁路，即在满足这些体内要求的条件下，两个主要的紫外线诱导性病变和由化学致癌物（G-AAF）形成的鸟嘌呤加合物。两个辅因子对于有效的Pol V介导的病变旁路至关重要：（i）稳定装有β钳的DNA底物; （ii）在病变部位下游组装的延伸的单链RecA / ATP细丝。为了有效旁路，Pol V需要同时与Beta钳和RecA灯丝的3'末端相互作用。在长的单链环状DNA模板上，最佳实现了延伸的RecA / ATP细丝的形成和稳定的β-钳位。与以前发布的数据相比，Pol V介导的病变旁路并非绝对需要单链DNA结合蛋白（SSB），只要ATP代替ATPgammaS激活RecA细丝即可。通过使用不适当的DNA底物或UmuC融合蛋白代替天然Pol V可以解释与现有文献的进一步差异。

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