Shibutani S, Fernandes A, Suzuki N, Zhou L, Johnson F, Grollman A P
Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651, USA.
J Biol Chem. 1999 Sep 24;274(39):27433-8. doi: 10.1074/jbc.274.39.27433.
Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.
位点特异性修饰的寡脱氧核苷酸被用于研究一种主要的烹饪食物诱变剂衍生的DNA加合物，即N-(脱氧鸟苷-8-基)-2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶(dG-C8-PhIP)的诱变特性。通过使含有单个dG(5'-TCCTCCTXGCCTCTC，其中X = C、A、G或T)的寡脱氧核苷酸与N-乙酰氧基-PhIP反应来制备dG-C8-PhIP修饰的寡脱氧核苷酸。将未修饰的和dG-C8-PhIP修饰的寡聚物插入单链噬菌粒载体中。这些单链载体被转染到猴肾(COS-7)细胞中。获得的子代质粒用于转化大肠杆菌DH10B。当dC位于dG-C8-PhIP的5'侧翼位置时，在dG-C8-PhIP相对位置观察到优先掺入正确的碱基dCMP。检测到靶向的G→T颠换，以及少量的G→A转换和G→C颠换。未修饰的载体未检测到突变。还探讨了序列背景对dG-C8-PhIP突变频率和谱的影响。当dC的5'侧翼碱基被dT、dA或dG取代时，突变谱与dC侧翼碱基时观察到的相似。当dC或dG位于dG-C8-PhIP的5'位置时，观察到更高的突变频率(28-30%)。当dA位于损伤的5'位置时，观察到较低的突变频率(13%)。仅当加合物侧翼为dG或dT时检测到单碱基缺失。我们得出结论，dG-C8-PhIP具有诱变作用，在哺乳动物细胞中主要产生G→T颠换。dG-C8-PhIP的突变频率和特异性取决于相邻的序列背景。
