Zhang Xuemei, Donnelly Alison, Lee Irene, Berdis Anthony J
Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Biochemistry. 2006 Nov 7;45(44):13293-303. doi: 10.1021/bi060418v.
Translesion DNA synthesis represents the ability of a DNA polymerase to misinsert a nucleotide opposite a DNA lesion. Previous kinetic studies of the bacteriophage T4 DNA polymerase using a series of non-natural nucleotides suggest that pi-electron density of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site, a nontemplating DNA lesion. However, it is surprising that these nonhydrogen-bonding analogues can also be incorporated opposite natural templating DNA with variable degrees of efficiency. In this article, we describe attempts to achieve selectivity for incorporation opposite the abasic site through optimization of pi-electron density and shape of the nucleobase. Toward this goal, we report the synthesis and enzymatic characterization of two novel nucleotide analogues, 5-napthyl-indolyl-2'-deoxyriboside triphosphate (5-NapITP) and 5-anthracene-indolyl-2'-deoxyriboside triphosphate (5-AnITP). The overall catalytic efficiency for their incorporation opposite an abasic site is similar to that of other non-natural nucleotides such as 5-NITP and 5-PhITP that contain significant pi-electron density. As expected, the incorporation of either 5-NapITP or 5-AnITP opposite templating DNA is reduced and presumably reflects steric constraints imposed by the large size of the polycyclic aromatic moieties. Furthermore, 5-NapITP is a chain terminator of translesion DNA synthesis because the DNA polymerase is unable to extend beyond the incorporated non-natural nucleotide. In addition, idle turnover measurements confirm that 5-NapIMP is stably incorporated opposite damaged DNA, and this enhancement reflects the overall favorable incorporation kinetic parameters coupled with a reduction in excision by the exonuclease-proofreading activity of the enzyme. On the basis of these data, we provide a comprehensive assessment of the potential role of pi-electron surface area for nucleotide incorporation opposite templating and nontemplating DNA catalyzed by the bacteriophage T4 DNA polymerase.
跨损伤DNA合成代表了DNA聚合酶在DNA损伤位点对面错误插入核苷酸的能力。先前使用一系列非天然核苷酸对噬菌体T4 DNA聚合酶进行的动力学研究表明，进入核苷酸的π电子密度对在无碱基位点（一种非模板化DNA损伤）对面进行掺入的效率有很大贡献。然而，令人惊讶的是，这些非氢键类似物也能以不同程度的效率在天然模板化DNA对面掺入。在本文中，我们描述了通过优化核碱基的π电子密度和形状来实现对无碱基位点对面掺入的选择性的尝试。为了实现这一目标，我们报告了两种新型核苷酸类似物5-萘基-吲哚基-2'-脱氧核糖核苷三磷酸（5-NapITP）和5-蒽基-吲哚基-2'-脱氧核糖核苷三磷酸（5-AnITP）的合成及酶学特性。它们在无碱基位点对面掺入的总体催化效率与其他含有显著π电子密度的非天然核苷酸如5-NITP和5-PhITP相似。正如预期的那样，5-NapITP或5-AnITP在模板化DNA对面的掺入减少，这可能反映了多环芳基部分的大尺寸所带来的空间位阻。此外，5-NapITP是跨损伤DNA合成的链终止剂，因为DNA聚合酶无法延伸超过掺入的非天然核苷酸。此外，空载周转率测量证实5-NapIMP稳定地掺入受损DNA对面，这种增强反映了总体有利的掺入动力学参数以及该酶的外切核酸酶校对活性导致的切除减少。基于这些数据，我们对噬菌体T4 DNA聚合酶催化的在模板化和非模板化DNA对面进行核苷酸掺入时π电子表面积的潜在作用进行了全面评估。
