title: 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.
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Bifeng
authors.initials: B
authors.lastname: Yuan
authors.forename: Jianshuang
authors.initials: J
authors.lastname: Wang
authors.forename: Huachuan
authors.initials: H
authors.lastname: Cao
authors.forename: Ruobai
authors.initials: R
authors.lastname: Sun
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D002853:Chromatography, Liquid; D004249:DNA Damage; D004261:DNA Replication; D059014:High-Throughput Nucleotide Sequencing; D016296:Mutagenesis; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
chemical_list: D009838:Oligodeoxyribonucleotides