title: A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis.
abstract: Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis-syn thymidine-thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.
authors.affiliation: Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Taggart
authors.forename: Terry L
authors.initials: TL
authors.lastname: Camerlengo
authors.forename: Jason K
authors.initials: JK
authors.lastname: Harrison
authors.forename: Shanen M
authors.initials: SM
authors.lastname: Sherrer
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Kshetry
authors.forename: John-Stephen
authors.initials: JS
authors.lastname: Taylor
authors.forename: Kun
authors.initials: K
authors.lastname: Huang
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016296:Mutagenesis; D011740:Pyrimidine Dimers / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D000094769:DNA Polymerase iota; D000099270:Y-Family DNA Polymerases
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011740:Pyrimidine Dimers; D000094769:DNA Polymerase iota; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein; C000722070:POLI protein, human