title: Mechanistic studies of the bypass of a bulky single-base lesion catalyzed by a Y-family DNA polymerase.
abstract: 1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dGAP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.
authors.affiliation: Department of Biochemistrythe Ohio State Biochemistry Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Shanen M
authors.initials: SM
authors.lastname: Sherrer
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Brown
authors.forename: Lindsey R
authors.initials: LR
authors.lastname: Pack
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Jasti
authors.forename: Jason D
authors.initials: JD
authors.lastname: Fowler
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D019641:DNA, Archaeal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011721:Pyrenes / Q000737:chemistry*; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D001335:Vehicle Emissions
chemical_list: D019843:Archaeal Proteins; D018736:DNA Adducts; D019641:DNA, Archaeal; D011721:Pyrenes; D001335:Vehicle Emissions; D004259:DNA-Directed DNA Polymerase; C032668:1-nitropyrene