Correct and incorrect nucleotide incorporation pathways in DNA polymerase beta.
Correct and incorrect nucleotide incorporation pathways in DNA polymerase beta.
复制标题
DNA 聚合酶 beta 中正确和错误的核苷酸掺入途径。
DOI：
10.1016/j.bbrc.2006.09.059
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发表时间：
2006
影响因子：
3.1
通讯作者：
Schlick,Tamar
中科院分区：
文献类型：
--
作者：
Radhakrishnan,Ravi;Schlick,Tamar
跟踪DNA复制和修复途径的结构和能量变化是理解这些重要过程的核心。在这里，我们报告了聚合酶催化的磷酸转移反应的有利机制，对应于DNA中正确和不正确的核苷酸结合，使用了一种新的协议，涉及能量最小化、动力学模拟、准谐自由能计算和量子力学/分子力学混合动力学模拟。尽管所提出的途径可能不是唯一的并且会引起不同的变化，但几何和能量论点支持本文所揭示的G：C和G：A体系中磷酸转移途径中的一系列瞬时中间体，该过程涉及水分子与Pollβ‘S活性中心中三个保守的天冬氨酸残基之间的质子转移的格洛图斯跳跃机制。在G：C系统中，限速步骤是初始质子跃迁，其自由活化能至少为17kcal/mol，这与测量的kpol值非常接近。PolDNA的保真度鉴别可以解释为G：A系统中酶的闭合三元络合物的稳定性显著丧失，以及亲核攻击的初始步骤，即末端β引物O_3‘H基团的去质子化的激活能大大提高。因此，匹配和不匹配的碱基对之间酶活性位点的细微差异会导致催化性能的显著差异。
Tracking the structural and energetic changes in the pathways of DNA replication and repair is central to the understanding of these important processes. Here we report favorable mechanisms of the polymerase-catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA by using a novel protocol involving energy minimizations, dynamics simulations, quasi-harmonic free energy calculations, and mixed quantum mechanics/molecular mechanics dynamics simulations. Though the pathway proposed may not be unique and invites variations, geometric and energetic arguments support the series of transient intermediates in the phosphoryl transfer pathways uncovered here for both the G:C and G:A systems involving a Grotthuss hopping mechanism of proton transfer between water molecules and the three conserved aspartate residues in pol β’s active-site. In the G:C system, the rate-limiting step is the initial proton hop with a free energy of activation of at least 17kcal/mol, which corresponds closely to measured kpolvalues. Fidelity discrimination in pol β can be explained by a significant loss of stability of the closed ternary complex of the enzyme in the G:A system and much higher activation energy of the initial step of nucleophilic attack, namely deprotonation of terminal DNA primer O3′H group. Thus, subtle differences in the enzyme active-site between matched and mismatched base pairs generate significant differences in catalytic performance.