Evaluating the contribution of base stacking during translesion DNA replication
Evaluating the contribution of base stacking during translesion DNA replication
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DOI：
10.1021/bi034948s
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发表时间：
2004-01-20
期刊：
影响因子：
2.9
通讯作者：
Berdis, AJ
中科院分区：
文献类型：
--
作者：
Reineks, EZ;Berdis, AJ
尽管基本部位具有非模板性，DAMP通常优先插入病变的对面，这一现象通常被称为“A规则”。我们已经使用彻底的动力学方法来评估跨损伤DNA复制过程中的聚合效率，从而评估了导致这一独特行为的分子机制。使用噬菌体T4DNA聚合酶，我们已经测量了一系列修饰核苷酸的插入，并证明了增加碱基的大小与相对于基本位置的插入效率的增加没有相关性。一种类似物，5-硝基吲哚-2‘-脱氧核苷三磷酸，是独一无二的，因为它被插入到病变对面，与湿插入相比，其效率大约高出1000倍。稳态前动力学测量得到5-硝基吲哚-2‘-脱氧核糖苷三磷酸与碱基相对插入时的k(pol值)为126kol(-1)，K-d值为18um。这些数值与酶促形成天然沃森-克里克碱基对的数值不相上下。这些结果不仅重申了氢键对于核苷酸插入是不必要的，而且也表明了进入的核苷酸碱基的碱基堆积和/或去溶解能力确实在产生高效的DNA聚合中起着主导作用。提供了一种解释这种独特的核苷酸碱基催化效率提高的模型，并调用了传入核苷酸碱基的芳香族部分与聚合酶活性部位中存在的芳香族氨基酸的pi-pi堆积相互作用。最后，在噬菌体T4DNA聚合酶和Klenow片段之间，测量了5-硝基吲哚-2‘-脱氧核苷三磷酸插入碱性位点的速率的差异。这些动力学差异被解释为两种酶之间各种结构成分的差异，并与所提出的DNA聚合模型一致。
Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule". We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication. Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site. One analogue, 5-nitroindolyl- 2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion. Pre-steady-state kinetic measurements yield a k(pol) value of 126 s(-1) and a K-d value of 18 muM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site. These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair. These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization. A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site. Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment. These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.