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Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction
Nature Chemical Biology ( IF 14.8 ) Pub Date : 2017-07-31 00:00:00 , DOI: 10.1038/nchembio.2450
David D Shock , Bret D Freudenthal , William A Beard , Samuel H Wilson

DNA polymerases catalyze efficient and high-fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, that removes the DNA primer terminus and generates deoxynucleoside triphosphates. Because pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that this reaction was limited by a nonchemical step. Use of a pyrophosphate analog, in which the bridging oxygen is replaced with an imido group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect, indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium favoring the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.

中文翻译:

调节DNA聚合酶β反应平衡以分解逆反应

DNA聚合酶催化有效和高保真的DNA合成。尽管该反应有利于核苷酸掺入,但是聚合酶还催化逆反应,即焦磷酸解,其去除了DNA引物末端并生成了脱氧核苷三磷酸。由于焦磷酸解会影响聚合酶的保真度和对链终止核苷的敏感性,因此我们用人DNA聚合酶β分析焦磷酸解,发现该反应效率低下。缺少硫代元素作用表明该反应受到非化学步骤的限制。焦磷酸酯类似物的使用,其中桥连的氧被亚氨基(PNP)取代,增加了逆反应的速率,并显示出较大的硫代元素效应,表明化学反应现在在确定速率。具有PNP的延时晶体学捕获的结构与有利于逆反应的化学平衡相一致。这些结果凸显了在β-和反应化学,酶构象变化和整体反应平衡中传入核苷酸的γ-磷酸盐。
更新日期:2017-09-20
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