title: Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase.
abstract: 3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-N-ABA)). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG(C8-N-ABA) is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG(C8-N-ABA) on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG(C8-N-ABA) lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG(C8-N-ABA) lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG(C8-N-ABA) bypass catalyzed by Dpo4.
authors.affiliation: The Ohio State Biochemistry Program, Columbus, OH 43210, USA; Department of Chemistry and Biochemistry, Columbus, OH 43210, USA.
authors.forename: Varun V
authors.initials: VV
authors.lastname: Gadkari
authors.affiliation: Department of Chemistry and Biochemistry, Columbus, OH 43210, USA; The Ohio State University Biophysics Program, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: E John
authors.initials: EJ
authors.lastname: Tokarsky
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Chanchal K
authors.initials: CK
authors.lastname: Malik
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
authors.affiliation: The Ohio State Biochemistry Program, Columbus, OH 43210, USA; Department of Chemistry and Biochemistry, Columbus, OH 43210, USA; The Ohio State University Biophysics Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D000393:Air Pollutants / Q000633:toxicity; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001551:Benz(a)Anthracenes / Q000633:toxicity; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics
chemical_list: D000393:Air Pollutants; D001426:Bacterial Proteins; D001551:Benz(a)Anthracenes; D018736:DNA Adducts; D004269:DNA, Bacterial; D019951:DNA Polymerase beta; C117220:3-nitrobenzanthrone
keywords: 3-Nitrobenzanthrone; Mutation; NitroPAH; Pre-steady-state kinetics; Sulfolobus solfataricus Dpo4; Y-family DNA polymerase