Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Nucleic Acids Res. 2024 Jul 22;52(13):7437-7446. doi: 10.1093/nar/gkae529.
Formamidopyrimidine (Fapy•dG) is a major lesion arising from oxidation of dG that is produced from a common chemical precursor of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). In human cells, replication of single-stranded shuttle vectors containing Fapy•dG is more mutagenic than 8-OxodGuo. Here, we present the first data regarding promoter dependent RNA polymerase II bypass of Fapy•dG. 8-OxodGuo bypass was examined side-by-side. Experiments were carried out using double-stranded shuttle vectors in HeLa cell nuclear lysates and in HEK 293T cells. The lesions do not significantly block transcriptional bypass efficiency. Less than 2% adenosine incorporation occurred in cells when the lesions were base paired with dC. Inhibiting base excision repair in HEK 293T cells significantly increased adenosine incorporation, particularly from Fapy•dG:dC bypass which yielded ∼25% adenosine incorporation. No effect was detected upon transcriptional bypass of either lesion in nucleotide excision repair deficient cells. Transcriptional mutagenesis was significantly higher when shuttle vectors containing dA opposite one of the lesions were employed. For Fapy•dG:dA bypass, adenosine incorporation was greater than 85%; whereas 8-OxodGuo:dA yielded >20% point mutations. The combination of more frequent replication mistakes and greater error-prone Pol II bypass suggest that Fapy•dG is more mutagenic than 8-OxodGuo.
甲酰胺嘧啶（Fapy•dG）是一种主要的损伤，它是由 8-氧代-7,8-二氢-2'-脱氧鸟苷（8-OxodGuo）的常见化学前体产生的 dG 氧化而来。在人类细胞中，含有 Fapy•dG 的单链穿梭载体的复制比 8-OxodGuo 更具突变性。在这里，我们首次提供了关于依赖启动子的 RNA 聚合酶 II 绕过 Fapy•dG 的数据。同时检查了 8-OxodGuo 的绕过情况。实验是在 HeLa 细胞核裂解物和 HEK 293T 细胞中使用双链穿梭载体进行的。这些损伤不会显著阻断转录旁路效率。当损伤与 dC 碱基配对时，细胞中不到 2%的腺嘌呤掺入。在 HEK 293T 细胞中抑制碱基切除修复显著增加了腺嘌呤掺入，特别是 Fapy•dG:dC 旁路的掺入率约为 25%。在核苷酸切除修复缺陷细胞中，两种损伤的转录旁路均未检测到影响。当使用含有一个损伤的 dA 对面的穿梭载体时，转录突变明显更高。对于 Fapy•dG:dA 旁路，腺嘌呤掺入大于 85%；而 8-OxodGuo:dA 产生超过 20%的点突变。更频繁的复制错误和更易错的 Pol II 旁路的结合表明，Fapy•dG 比 8-OxodGuo 更具突变性。
