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 N2,3-ethenoguanine (N2,3-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 N2,3-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 N2,3-εG and its isomer 1,N2-ethenoguanine (1,N2-εG) were evaluated in various repair and replication backgrounds. We found that N2,3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N2-εG induces various substitutions and frameshifts. We also found that N2,3-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 N2,3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.

During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase β replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.5-chloro-deoxycytidine | hypochlorite | myeloperoxidase | inflammatory bowel disease | carcinogenesis

We report the x-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, a target for anti-cancer drugs. Bisphosphonates containing unbranched sidechains bind to either the farnesyl diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg 2+ that occupy the same position as found in FPP synthase. However, each of three "V-shaped" bisphosphonates binds to both the FPP and GGPP sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an RMS error of 1.3Å. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2x, using a comparative molecular similarity analysis, based on a docked-structure alignment. These results show that some GGPPS inhibitors can occupy both substrate and product site, and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anti-cancer agents.