Reineks Edmunds Z, Berdis Anthony J
Department of Pharmacology and the Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Biochemistry. 2004 Jan 20;43(2):393-404. doi: 10.1021/bi034948s.
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 kpol value of 126 s(-1) and a Kd value of 18 microM 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.
尽管无碱基位点不依赖模板，但脱氧腺苷一磷酸（dAMP）常常优先插入到损伤位点的对面，这种现象通常被称为“ A规则”。我们使用一种全面的动力学方法来评估跨损伤DNA复制过程中的聚合效率，从而评估了导致这种独特行为的分子机制。利用噬菌体T4 DNA聚合酶，我们测量了一系列修饰核苷酸的插入情况，并证明增加核碱基的大小与在无碱基位点对面的插入效率增加无关。一种类似物，5-硝基吲哚-2'-脱氧核糖核苷三磷酸，很独特，因为它插入到损伤位点对面的效率比dAMP插入的效率高约1000倍。稳态前动力学测量得出，5-硝基吲哚-2'-脱氧核糖核苷三磷酸插入无碱基位点对面时的聚合酶催化常数（kpol）值为126 s(-1)，解离常数（Kd）值为18 microM。这些值与天然沃森-克里克碱基对的酶促形成相关的值相当。这些结果不仅重申了氢键对于核苷酸插入不是必需的，而且还表明进入的核碱基的碱基堆积和/或去溶剂化能力可能确实在产生高效DNA聚合中起主要作用。提供了一个解释这种独特核碱基催化效率增加的模型，该模型涉及进入的核碱基的芳香部分与聚合酶活性位点中存在的芳香族氨基酸之间的π-π堆积相互作用。最后，测量了噬菌体T4 DNA聚合酶和klenow片段在无碱基位点对面插入5-硝基吲哚-2'-脱氧核糖核苷三磷酸的速率差异。这些动力学差异根据两种酶之间各种结构成分的差异进行了解释，并且与提出的DNA聚合模型一致。
