Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy<middle dot>dG

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<middle dot>dG), are produced from a common chemical intermediate. Fapy<middle dot>dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy<middle dot>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<middle dot>dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy<middle dot>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<middle dot>dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy<middle dot>dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy<middle dot>dG:C base pair. The alpha- and beta-configurational isomers of Fapy<middle dot>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<middle dot>dG, five different structures were solved, including Fapy<middle dot>dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy<middle dot>dG:C product state (postchemistry), and error-prone Fapy<middle dot>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<middle dot>dG lesions impact transcription and subsequent pathological consequences.