title: Translesion synthesis across polycyclic aromatic hydrocarbon diol epoxide adducts of deoxyadenosine by Sulfolobus solfataricus DNA polymerase Dpo4.
abstract: The mechanisms by which derivatives of polycyclic aromatic hydrocarbons (PAHs) cause mutations have been of considerable interest. Three different N(6)-adenyl PAH-diol epoxide oligonucleotide derivatives were studied with the archebacterial translesion DNA polymerase Sulfolobus solfataricus Dpo4. Steady-state kinetic analysis indicated insertion of all four dNTPs opposite each of the three N(6)-adenyl PAH adducts, with only slightly varying misincorporation efficiencies. Full-length extension of shorter primers paired with templates containing the N(6)-adenyl PAH derivatives proceeded to apparent completion at 45 degrees C in the presence of added dimethyl sulfoxide. Analysis of the products by high-performance liquid chromatography/collision-induced mass spectrometry indicated the presence of mixtures of products with each PAH adduct. These mixtures correspond to both error-free synthesis and mixtures of polymerization/realignment steps. With an unmodified template, only the expected A:T and G:C pairing was detected in the primer extension products under these conditions, with no frameshifts. These results demonstrate the complexity of polymerization opposite these bulky N(6)-adenyl PAH adducts, even with a single polymerase.
authors.affiliation: Department of Biochemistry and Chemistry, Vanderbilt University, Nashville, Tennessee 37232-0146, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Zang
authors.forename: Goutam
authors.initials: G
authors.lastname: Chowdhury
authors.forename: Karen C
authors.initials: KC
authors.lastname: Angel
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Harris
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry* / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry* / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D015394:Molecular Structure; D009841:Oligonucleotides / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry* / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature
chemical_list: D003839:Deoxyadenosines; D003854:Deoxyribonucleotides; D004852:Epoxy Compounds; D009841:Oligonucleotides; D011084:Polycyclic Aromatic Hydrocarbons; D004259:DNA-Directed DNA Polymerase