PDF(Sci-hub)
Abstract：
O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5\'-AATAGTATCT[O(6)-POB-G]GAGCC-3\') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).
摘要：
O（6）-POB-dG（O（6）-[4-氧代-4-（3-吡啶基）丁-1-基]脱氧鸟苷）是由代谢激活的烟草特异性亚硝胺如4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）和N-亚硝基烟碱（NNK）。如果不修理,O（6）-POB-dG加合物在DNA复制过程中引起错配，导致G→A和G→T突变。一种专门的DNA修复蛋白，O(6)-烷基鸟嘌呤-DNA-烷基转移酶(AGT),将POB基团从DNA中的O（6）-POB-dG转移到蛋白质中的半胱氨酸残基（Cys145），从而恢复正常的鸟嘌呤和防止诱变。AGT介导的O(6)-POB-dG修复率可能受局部DNA序列环境的影响,可能导致加合物积累和在基因组内的特定位点增加的诱变。在目前的工作中,开发了基于同位素稀释的高效液相色谱-电喷雾电离串联质谱（HPLC-ESI（）-MS/MS）的方法，以研究DNA序列对AGT介导的O（6）修复动力学的影响-POB-dG加合物。在我们的方法中，将在指定位点含有O(6)-POB-dG的合成DNA双链体与重组人AGT蛋白一起孵育确定的时间段。用D（4）-O（6）-POB-dG内标加标并温和酸水解后释放O（6）-POB-鸟嘌呤（O（6）-POB-G）和D（4）-O（6）-POB-鸟嘌呤（D（4）-O（6）-POB-G），样品通过固相萃取（SPE）纯化，DNA中残留的O(6)-POB-G加合物通过毛细管HPLC-ESI(+)-MS/MS定量。通过分析包含已知量的含O（6）-POB-G的DNA和相应的未修饰的DNA双链体的混合物，并通过检查在有增加量的AGT蛋白存在下烷基转移的动力学来验证新方法。通过胰蛋白酶肽的HPLC-ESI（）-MS/MS测定，O（6）-POB-dG从DNA中消失伴随着AGTCys-145的吡啶基氧基丁基化。通过确定位于代表修饰的大鼠H-ras序列（5'-AATAGTATCT[O（6）-POB-G]GAGCC-3\'）的DNA双链体中的AGT介导的O（6）-POB-dG加合物修复的二级动力学，证明了新方法的适用性。当O（6）-POB-1与10配对时，观察到烷基转移速率较快（-1）（
