The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited
The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited
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DOI：
10.1016/j.jmb.2004.06.017
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发表时间：
2004-08-06
影响因子：
5.6
通讯作者：
Fuchs, RP
中科院分区：
文献类型：
--
作者：
Fujii, S;Gasser, W;Fuchs, RP
除了复制型DNA聚合酶，细胞还含有参与损伤耐受性、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）参与跨损伤合成（TLS）和诱变。遗传研究已经确定诱变需要UmuC和UmuD的蛋白水解产物（UmuD '）。此外，RecA蛋白和复制持续性因子β-钳夹在遗传学上被发现是诱变的必要辅助因子。在这里，我们重建了三个常见的复制阻断病变，即两个主要的UV诱导的病变和鸟嘌呤加合物形成的化学致癌物（G-AAF）的条件下，满足这些在体内的要求，PolV介导的旁路。两种辅因子对于有效的Pol V介导的病变绕过是必需的：（i）其上稳定加载有β-夹的DNA底物;和（ii）从病变位点下游组装的延伸的单链RecA/ATP细丝。为了有效的旁路，Pol V需要同时与β-钳和RecA细丝的3'末端相互作用。在长的单链环状DNA模板上最好地实现延伸的RecA/ATP细丝的形成和β-夹的稳定加载。与先前发表的数据相反，单链DNA结合蛋白（SSB）不是Pol V介导的病变旁路所绝对需要的，只要ATP而不是ATP γ S激活RecA细丝。与现有文献的进一步差异可通过使用不充分的DNA底物或UmuC融合蛋白而不是天然Pol V（C）2004 Elsevier Ltd.来解释。保留所有权利。
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 (C) 2004 Elsevier Ltd. All rights reserved.