title: Comparison of the in vitro replication of the 7-(2-oxoheptyl)-1,N2-etheno-2'-deoxyguanosine and 1,N2-etheno-2'-deoxyguanosine lesions by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4).
abstract: Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to that of the 1,N(2)-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in 5'-TXG-3' local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5'-CXG-3' local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N(2)-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5'-TXG-3' sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pK(a) for this transition was lower than that observed for the 1,N(2)-epsilon-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pK(a) value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N(2)-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.
authors.affiliation: Department of Chemistry, Department of Biochemistry, and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235-1822, USA.
authors.forename: Plamen P
authors.initials: PP
authors.lastname: Christov
authors.forename: Katya V
authors.initials: KV
authors.lastname: Petrova
authors.forename: Ganesh
authors.initials: G
authors.lastname: Shanmugam
authors.forename: Ivan D
authors.initials: ID
authors.lastname: Kozekov
authors.forename: Albena
authors.initials: A
authors.lastname: Kozekova
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.forename: Michael P
authors.initials: MP
authors.lastname: Stone
authors.forename: Carmelo J
authors.initials: CJ
authors.lastname: Rizzo
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019951:DNA Polymerase beta / Q000378:metabolism*; D003839:Deoxyadenosines / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009841:Oligonucleotides / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: C555804:7-(2-oxoheptyl)-1,N2-etheno-2'-deoxyguanine; D003839:Deoxyadenosines; D009841:Oligonucleotides; D006147:Guanine; C032221:1,N(6)-ethenodeoxyadenosine; D019951:DNA Polymerase beta