Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, USA.
Division of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA.
J Biol Chem. 2024 Sep;300(9):107719. doi: 10.1016/j.jbc.2024.107719. Epub 2024 Aug 29.
Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) are major products of 2'-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer-dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to 2'-deoxyguanosine. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G, or U. Fitting of reaction data indicates that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast- or slow-reacting base pairs vary less than twofold as a function of the nucleotide opposite the lesion. Promoter-dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side by side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.
Fapy•dG（N6-（2-脱氧-α，β-D-赤戊呋喃糖基）-2,6-二氨基-4-羟基-5-甲酰胺嘧啶）和 8-OxodGuo（8-氧代-7,8-二氢-2'-脱氧鸟苷）是 2'-脱氧鸟苷氧化的主要产物。Fapy•dG 的不同寻常之处在于它作为一个动态混合物的异构体存在。关于 Fapy•dG 对转录旁路的影响，人们了解得要少得多。这里呈现的数据表明，T7 RNA 聚合酶（T7 RNAP）对 Fapy•dG 的旁路比 8-OxodGuo 更复杂。与 2'-脱氧鸟苷相比，T7 RNAP 对 Fapy•dG 的引物依赖性转录旁路受到阻碍。T7 RNAP 在 miniscaffold 中掺入胞嘧啶与 Fapy•dG 配对的速度比 A、G 或 U 快至少 13 倍。反应数据的拟合表明，Fapy•dG 的异构体在动力学上是可区分的。在 Fapy•dG 之后延伸新生转录物的能力弱取决于与损伤相对的核苷酸。描述延伸通过快速或慢速反应碱基对的速率常数作为与损伤相对的核苷酸的函数变化不到两倍。进行了启动子依赖性 T7 RNAP 对 Fapy•dG 和 8-OxodGuo 的旁路，同时进行。8-OxodGuo 旁路导致超过 55%的 A 与之相对。当穿梭载体包含 Fapy•dG:dA 碱基对时，在 Fapy•dG 旁路后，会产生高达 20%的点突变和 9%的单核苷酸缺失。Fapy•dG:dC 碱基对易错旁路约占转录物的 9%。在 RNA 产物中测量的 Fapy•dG 和 8-OxodGuo 的转录旁路突变频率与复制错误相当或大于复制错误，这表明这些损伤可能通过转录显著导致突变。
