Division of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, CT 06269, U.S.A.
Biochem J. 2023 May 15;480(9):649-664. doi: 10.1042/BCJ20230029.
Inosine is a key intermediate in de novo purine nucleotide biosynthesis in cells. Inosine is known to be mutagenic when it is present in DNA, in place of adenine via deamination, by facilitating the incorporation of dCTP exclusively, resulting in A:T to G:C mutation. The structural basis for the mutagenicity of inosine bypass has been reported in some DNA polymerases including human DNA polymerase eta (polη). However, the structural and biochemical basis for the mutagenic potential of the incorporation of deoxyinosine triphosphate (dITP) into DNA remains poorly understood. To gain insights into the mutagenic potential of the incorporation of inosine into DNA, we conducted structural and kinetic studies of human polη incorporating dITP across undamaged DNA template containing dC or dT. Polη incorporated dITP opposite dC 14-fold more efficiently than opposite dT, indicating that dITP incorporation by polη can be mutagenic unlike the bypass of inosine by polη, which incorporated dCTP almost exclusively opposite the templating inosine over dTTP (70:1). Polη-dC:dITP crystal structure showed that the incoming dITP formed Watson-Crick base pair along with wobble base pair via 4-imino-2-keto tautomer of cytosine diminishing the catalytic efficiency compared to dGTP incorporation across dC. In addition, the crystal structure of polη-dT:dITP revealed that dT and dITP formed Watson-Crick like base pair via 4-enol-2-keto tautomer of thymine, reinforced by wobble base pair via 4-keto-2-keto tautomer of thymine resulting in the increased mutagenicity of dITP incorporation (14:1 across dC and dT), which is 14-fold higher than dGTP incorporation by polη (190:1 across dC and dT).
肌苷是细胞中新嘌呤核苷酸合成的关键中间产物。肌苷在 DNA 中存在时，通过脱氨作用取代腺嘌呤，特别有利于 dCTP 的掺入，导致 A:T 到 G:C 突变，被认为是诱变剂。一些 DNA 聚合酶，包括人类 DNA 聚合酶 η（polη），已经报道了肌苷旁路的诱变机制的结构基础。然而，脱氧肌苷三磷酸（dITP）掺入 DNA 的诱变潜力的结构和生化基础仍知之甚少。为了深入了解肌苷掺入 DNA 的诱变潜力，我们对人类 polη 在包含 dC 或 dT 的未受损 DNA 模板上掺入 dITP 进行了结构和动力学研究。polη 掺入 dITP 与 dC 的效率比与 dT 的效率高 14 倍，表明 polη 掺入 dITP 具有诱变作用，而 polη 旁路肌苷时几乎只优先掺入 dCTP 而不是 dTTP（70:1）。polη-dC:dITP 晶体结构表明，与 dGTP 掺入 dC 相比，进入的 dITP 通过胞嘧啶的 4-亚氨基-2-酮互变异构体形成 Watson-Crick 碱基对和摆动碱基对，降低了催化效率。此外，polη-dT:dITP 的晶体结构表明，dT 和 dITP 通过胸腺嘧啶的 4-烯醇-2-酮互变异构体形成 Watson-Crick 样碱基对，通过胸腺嘧啶的 4-酮-2-酮互变异构体通过摆动碱基对得到加强，导致 dITP 掺入的诱变性增加（在 dC 和 dT 上为 14:1），比 polη 掺入 dGTP 的诱变性（在 dC 和 dT 上为 190:1）高 14 倍。
