PDF(Pubmed)
Abstract：
Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2\'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and β-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.
摘要：
氧化性DNA损伤对生物物种造成显著的有害影响。dG氧化导致的两个主要DNA损伤，8-氧代-7,8-二氢-2'-脱氧鸟苷（8-OxodGuo）和甲氨基嘧啶（Fapy·dG），是由常见的化学中间体生产的。在缺氧条件下以相当的产率形成Fapy·dG。Fapy·dG在人细胞中的复制旁路比8-OxodGuo更具诱变性。尽管转录诱变的生物学重要性，没有关于Fapy·dG对RNA聚合酶II（PolII）活性的影响的报道。在这里，我们进行全面的动力学研究，以调查Fapy·dG对PolII的三个关键转录保真度检查点步骤的影响：插入，扩展,和校对步骤。与未受损的dG相比，与Fapy·dG相反的无错误掺入与易出错掺入的比率显着降低。同样，Fapy·dG：错误对的扩展效率与无错误的扩展效率相当，Fapy·dG:C碱基对。Fapy·dG的α-和β-构型异构体对PolII的插入和延伸具有不同的影响。PolII可以通过校对优先切割容易出错的产品。为了进一步了解Fapy·dG转录过程的结构基础，解决了五种不同的结构，包括FAPY·DG模板加载状态(APO)，无差错三磷酸胞苷（CTP）结合状态（化学前），容易出错的ATP结合状态（化学前），无差错Fapy·dG：C产品状态（后化学），和容易出错的Fapy·dG：产品状态(后化学)，揭示独特的核苷酸结合和产物状态。一起来看,我们的研究为更好地理解Fapy·dG病变如何影响转录和随后的病理后果提供了一个全面的机制框架.
