Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides
Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides
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DOI：
10.1074/jbc.m112.366609
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发表时间：
2012-08-17
影响因子：
4.8
通讯作者：
Beese, Lorena S.
中科院分区：
文献类型：
--
作者：
Wang, Weina;Wu, Eugene Y.;Beese, Lorena S.
除了对碱基对错配具有辨别能力之外，DNA聚合酶对脱氧核糖核苷酸相对于核糖核苷酸或双脱氧核苷酸表现出高度的选择性。有人提出，一个活性位点残基（空间位阻门）会阻碍含有2'-羟基的核苷酸的有效结合。尽管这个空间位阻门在糖部分的辨别中起作用，但其相互作用并不能完全解释所观察到的突变体的行为。在此，我们展示了10个高分辨率的晶体结构以及对与脱氧核苷酸、核糖核苷酸和双脱氧核苷酸以及一个DNA底物复合的芽孢杆菌DNA聚合酶I大片段变体的酶动力学分析。综合来看，这些数据呈现了一种更细致和通用的核苷酸辨别机制，其中活性位点的中间构象集合会捕获非同源底物。已知活性位点的O - 螺旋在没有核苷酸底物时从开放状态转变为三元复合物的闭合状态，在闭合状态下反应基团排列整齐以进行催化。闭合状态下底物的错位在防止非同源核苷酸错误掺入方面起着根本作用。这里展示的结构表明，在开放和闭合状态这两个极端之间的额外O - 螺旋构象会产生一系列结合位点，这些位点会捕获并使非同源核苷酸错位。在完全闭合状态下不存在的水介导的相互作用在这些结合位点的形成中起着重要作用，并且可以被重塑以适应不同的非同源底物。这种机制可能也延伸到碱基对的辨别。
In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxy-ribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.