Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography
Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography
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DOI：
10.1016/j.jmb.2008.06.004
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发表时间：
2008-09-05
影响因子：
5.6
通讯作者：
Connolly, Bernard A.
中科院分区：
文献类型：
--
作者：
Firbank, Susan J.;Wardle, Josephine;Connolly, Bernard A.
古菌B族DNA聚合酶与模板链尿嘧啶紧密结合，并在遇到致突变前碱基时阻止复制。本文描述了在2.8埃分辨率下，与单链区域含有尿嘧啶的DNA引物模板复合物中的热球菌（Thermococcus gorgonarius）聚合酶的x射线晶体结构。DNA主干被扭曲，使尿嘧啶深深地位于聚合酶的氨基末端区域。特异性来自于蛋白质主链和尿嘧啶之间的氢键组合，其口袋形状是为了防止四个标准DNA碱基的稳定结合。与尿嘧啶两侧的两个磷酸盐有强烈的相互作用，该结构为尿嘧啶结合与停止复制的耦合提供了线索。关键氨基酸的重要性，通过结构分析和它们在古细菌聚合酶之间的保守性被确定，并通过位点定向诱变得到证实。V93Q是一种不再识别尿嘧啶的聚合酶变体，它的晶体结构也被报道，解释了V93Q的表型是通过尿嘧啶从口袋中被立体排斥。(C) 2008 Elsevier Ltd版权所有。
Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 angstrom resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer-template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket. (C) 2008 Elsevier Ltd. All rights reserved.