High-throughput analysis of the mutagenic and cytotoxic properties of DNA lesions by next-generation sequencing

Abstract

Human cells are constantly exposed to environmental and endogenous agents which can induce damage to DNA. Understanding the implications of these DNA modifications in the etiology of human diseases requires the examination about how these DNA lesions block DNA replication and induce mutations in cells. All previously reported shuttle vector-based methods for investigating the cytotoxic and mutagenic properties of DNA lesions in cells have low-throughput, where plasmids containing individual lesions are transfected into cells one lesion at a time and the products from the replication of individual lesions are analyzed separately. The advent of next-generation sequencing (NGS) technology has facilitated investigators to design scientific approaches that were previously not technically feasible or affordable. In this study, we developed a new method employing NGS, together with shuttle vector technology, to have a multiplexed and quantitative assessment of how DNA lesions perturb the efficiency and accuracy of DNA replication in cells. By using this method, we examined the replication of four carboxymethylated DNA lesions and two oxidatively induced bulky DNA lesions including (5′S) diastereomers of 8,5′-cyclo-2′-deoxyguanosine (cyclo-dG) and 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA) in five different strains of Escherichia coli cells. We further validated the results obtained from NGS using previously established methods. Taken together, the newly developed method provided a high-throughput and readily affordable method for assessing quantitatively how DNA lesions compromise the efficiency and fidelity of DNA replication in cells.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

“…This method enables the examination of the types and frequencies of lesion-induced errors generated by specific DNA polymerases. Although next-generation sequencing technology has previously been used to assess the mutagenic properties of carboxymethylated DNA lesions (22), only mutations opposite the lesion site were analyzed. In contrast, our design provides sequencing information for at least 10 template positions upstream and downstream from the DNA lesion site.…”

Generation of guanine–amino acid cross-links by a free radical combination mechanism

“…The cdA and cdG lesions have been found in γ-irradiated human cultured cells, 48, 49 and in humans. 50 The cyclopurine adducts are highly mutagenic 51, 52 and their accumulation in cellular DNA may contribute to the etiology of cancer and other diseases. 49, 53–55 The guanine – thymine tandem lesions, G[8-5m]T are formed by the addition of 5-(uracilyl) methyl radicals to the C8 position of guanine to form a radical adduct that is rapidly oxidized by traces of oxygen or other weak oxidants 56–60 .…”

The Application of Omics Technologies to the Study of Mammalian Toxicology

“…Identification of different patterns of mutagenesis may provide insight into causal mechanisms behind the mutagenic process [26,43,[132][133][134] These data suggested an odds ratio of approximately 26 for developing SJS in individuals that are carriers of the HLA-A*3101 allele. Considering the effect size of this variant, it would likely be of value clinically to perform genotyping of patients prior to the administration of carbamazepine in order to limit the risk of SJS.…”