Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland).
Chembiochem. 2013 Sep 2;14(13):1634-9. doi: 10.1002/cbic.201300296. Epub 2013 Aug 19.
O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.
O⁶-甲基鸟嘌呤（O⁶-MeG）是一种诱变 DNA 损伤，它是由 DNA 中鸟嘌呤（G）上的甲基化剂作用产生的。Dpo4 是一种参与跨损伤 DNA 合成（TLS）的古菌低保真 Y 家族 DNA 聚合酶，是研究人类 Y 家族聚合酶如何绕过 DNA 加合物的模型。先前的工作表明，Dpo4 介导的 dTTP 掺入优先发生在 O⁶-MeG 而不是 G 对面。然而，影响 Dpo4 对 O⁶-MeG 对面掺入 dTTP 的偏好的因素尚未完全确定。在这项研究中，我们研究了进入的 dNTP 的结构特征对其在 DNA 模板中对面 O⁶-MeG 掺入的酶促掺入的影响。为此，我们利用一种新的基于荧光的引物延伸测定法来评估 Dpo4 对面 G 或 O⁶-MeG 的一系列合成 dNTP 的掺入效率。在单 dNTP 引物延伸研究中，与 O⁶-MeG 相比，合成 dNTP 优先掺入 G 对面。此外，嘧啶基 dNTP 通常比嘌呤基 syn-构象 dNTP 更好地掺入。结果表明，进入的 dNTP 的疏水性似乎对 Dpo4 的核苷酸选择过程影响不大，氢键能力是主要影响因素。此外，dCTP 的 C2 位置的修饰增加了对面 O⁶-MeG 的掺入选择性，而效率没有明显损失。
