Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG

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 αand 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.

Promoter dependent RNA polymerase II bypass of the epimerizable DNA lesion, Fapy•dG and 8-Oxo-2′-deoxyguanosine

“…Structural data indicate that Fapy•dG predominantly adopts the β-configuration opposite dC; whereas the lesion exists as a dynamic mixture of anomers when opposed by dA, even in the crystalline state ( 32 ). Primer dependent Pol II kinetic data describing extension past a Fapy•dG:N (N = A, G or U) obtained using a miniscaffold also indicate that both anomers of the lesion are present ( 45 ). The relative bypass efficiencies can be attributed to this structural difference, provided Pol II is more tolerant of β-Fapy•dG in the template strand than α-Fapy•dG.…”

“…This possibility is supported by kinetic measurements carried out on a miniscaffold in which a mixture of α- and β-Fapy•dG is unpaired in a template. Pol II incorporates C opposite one of the anomers, presumably the β-isomer, more than 40-times faster than opposite the other isomer ( 45 ).…”

“…When Fapy•dG is paired with dA, the enzyme is likely to interact with roughly equal amounts of the α- and β-configurations of the lesion ( 32 ). Experiments in a miniscaffold indicate that Pol II incorporates A opposite one Fapy•dG anomer as rapidly as it does C ( 45 ). In contrast, Pol II greatly prefers to incorporate C opposite Fapy•dG when the lesion is paired with dC.…”

“…8-OxodGuo-induced transcriptional mutagenesis by Pol II has also been characterized in mammalian cells when the lesion is in the template strand or present as the corresponding nucleoside triphosphate ( 13 , 43 , 44 ). The effects of Fapy•dG on Pol II activity are limited to primer dependent kinetic analyses of transcriptional bypass within a miniscaffold that revealed the contribution of the α- and β-configurational isomers ( 45 ). Although Pol II inserts C opposite either Fapy•dG anomer more rapidly than A, G or U, all 4 nucleotides are incorporated, and the primer is extended past the lesion in the template.…”