The O 6-alkylguanosine adduct O 6-carboxymethyldeoxyguanosine (O 6-CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O 6-CMdG as well as the corresponding methyl adduct O 6-methyldeoxyguanosine (O 6-MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O 6-CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O 6-CMdG and O 6-MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O 6-serine-CMdG (O 6-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O 6-Ser-CMdG forms as an intermediate that spontaneously decomposes to form O 6-CMdG. Finally, we quantified levels of O 6-CMdG and O 6-MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.
O 6-carboxymethylguanine (O 6-CMG) is a mutagenic DNA adduct that forms at increased levels when people eat meat. It has been studied as a potential initiating event in colorectal carcinogenesis. It can arise from alkylation of guanine in DNA by electrophilic degradation products of N-nitroso compounds. There is significant data regarding biochemical and cellular process, including DNA repair and translesion DNA synthesis that control O 6-CMG accumulation, persistence, and mutagenicity. Mutation spectra arising from the adduct closely resemble common mutations in colorectal cancer; however, gaps remain in understanding the biochemical processes that regulate how and where the damage persists in the genome. Addressing such questions relies on advances in chemistry such as synthesis approaches and bioanalytical methods. Results of research in this area help advance our understanding of the toxicological relevance of O 6-CMG-modified DNA. Further attention should focus on understanding how a combination of genetic and environmental factors control its biological persistence and how this information can be used as a basis of biomoniotoring and prevention efforts to help mitigate colon cancer risk.
Carboxymethylation of DNA, including the formation of the DNA adduct O 6-carboxymethylguanine (O 6-CMG) is associated with lifestyle factors, such as diet. It can impede replicative polymerases (Pols) and lead to replication fork stalling, or an alternative means for replication to proceed by translesion DNA synthesis (TLS). TLS requires specialized DNA Pols characterized by open and preformed active sites capable of preferential bypass of alkylated DNA adducts, but that have high error rates, leading to mutations. Human TLS Pols can bypass O 6-CMG with varying degrees of accuracy, but it is not known how the chemical structure of the O 6-CMG adduct influences polymerase proficiency or fidelity. To better understand how adduct structure determines dNTP selection at lesion sites, we prepared DNA templates with a series of O 6-CMG structural analogs and compared the primer extension patterns of Y-and X-family Pols in response to these modifications. The results indicate that the structure of the DNA adduct had a striking effect on dNTP selection by Pol κ and that an increased steric size influences the fidelity of Pol η, whereas Pol ι and β function were only marginally affected. To test the hypothesis that specific hydrogen bonding interactions between the templating base and the incoming dNTP are a basis of this selection, we modeled the structural analogs with incoming dNTP in the Pol κ active site. These data indicate that the base pairing geometry and enhanced stabilization through a dense hydrogen bonding pattern are important molecular features in the dNTP incorporation fidelity of Pol κ. The study provides a basis for understanding error-free bypass of O 6-CMG by Pol κ and illustrates that the Pol relies strongly on hydrogen-bond complementarity for nucleotide selection.
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