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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D
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Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D

机译：细菌DNA连接酶D的聚合酶成分的原子结构和非同源末端连接功能

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摘要

DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribo-nucleotides to blunt DNA ends. Here we report the 1.5-A crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP/dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the pri-mase-like polymerase family in DNA repair.
机译：DNA连接酶D（LigD）是一种大型多功能蛋白质，它参与细菌中最近发现的非同源末端连接途径。 LigD由与聚合酶结构域（Pol）融合的ATP依赖性连接酶结构域和磷酸酯酶模块组成。 Pol活性因其对锰的依赖性，执行模板化和非模板化引物延伸反应的能力以及其向平端DNA末端添加核糖核苷酸的偏好而出类拔萃。在这里，我们报告假单胞菌LigD的Pol结构域的1.5-A晶体结构及其与锰和ATP / dATP底物的复合物，揭示了具有双金属机制的最小化聚合酶，其折叠程度与古细菌DNA引发酶相似。突变分析突出显示了活性位点中与功能相关的原子接触。尽管在共晶体中观察到了不同的核苷构象和ATP与dATP的接触，但功能分析表明ATP结合模式是dNMP和rNMP掺入的有效构象。我们发现，独特的分枝杆菌LigD突变可消除聚合酶活性，从而通过消除重组连接处的核苷酸插入而在体内增加了平末端双链断裂修复的保真度。因此，LigD Pol是体内诱变非同源末端连接的直接催化剂。我们的研究强调了pri-ase-like聚合酶家族在DNA修复中以前没有的作用。

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