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 s

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• 作者：Plamen P. Christov ; Katya V. Petrova ; Ganesh Shanmugam ; Ivan D. Kozekov ; Albena Kozekova ; F. Peter Guengerich ; Michael P. Stone ; Carmelo J. Rizzo
• 刊名：Chemical Research in Toxicology
• 出版年：2010
• 出版时间：August 16, 2010
• 年：2010
• 卷：23
• 期：8
• 页码：1330-1341
• 全文大小：546K
• 年卷期：v.23,no.8(August 16, 2010)
• ISSN：1520-5010

文摘

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,N²-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,N²-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,N²-ε-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,N²-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.