Next-generation sequencing reveals the biological significance of the N[superscript 2],3-ethenoguanine lesion in vivo

Abstract

Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N[superscript 2],3-ethenoguanine (N[superscript 2],3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2′-fluoro-2′-deoxyribose analog of N[superscript 2],3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N[superscript 2],3-εG and its isomer 1,N[superscript 2]-ethenoguanine (1,N[superscript 2]-εG) were evaluated in various repair and replication backgrounds. We found that N[superscript 2],3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N[superscript 2]-εG induces various substitutions and frameshifts. We also found that N[superscript 2],3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N[superscript 2],3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.