Generation of guanine-thymidine cross-links in DNA by peroxynitrite/carbon dioxide.
Generation of guanine-thymidine cross-links in DNA by peroxynitrite/carbon dioxide.
复制标题
通过过氧亚硝酸盐/二氧化碳在 DNA 中产生鸟嘌呤-胸苷交联。
DOI：
10.1021/tx200139c
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发表时间：
2011
影响因子：
4.1
通讯作者：
Shafirovich,Vladimir
中科院分区：
文献类型：
--
作者：
Yun,ByeongHwa;Geacintov,NicholasE;Shafirovich,Vladimir
过氧亚硝基碳酸酯是由二氧化碳和过氧亚硝基化合而成的一种重要的炎症介质。在水溶液中，它迅速分解为活性物质CO 3·-和·NO2自由基，已知这些自由基可以引发DNA中鸟嘌呤的选择性氧化和硝化。我们之前已经证明，碳酸根阴离子与2′-脱氧寡核糖核苷酸中鸟嘌呤的反应产生了一种以前未知的链内交联鸟嘌呤-胸腺嘧啶产物G*-T*，其C8（G*）和胸腺嘧啶N3（T*）原子之间具有共价键（CreanNucleic Acids Res. 2008，36，742−755）。在这项工作中，我们证明，G*-T* 交联产品也形成时，过氧亚硝酸盐（0.1 mM）在水溶液中与天然DNA反应（pH 7.5-7.7），含有25 mM二氧化碳/碳酸氢盐，此外还有熟知的鸟嘌呤硝化/氧化产物，如8-硝基鸟嘌呤（8-硝基-G）、5-胍基-4-硝基咪唑（NIm）、8-氧代-7，8-脱氢鸟嘌呤（8-氧代-G）和螺亚氨基二乙内酰脲（Sp）。用P1核酸酶和碱性磷酸酶酶解至核苷酸水平并经反相HPLC分离后，将这些产物的产率与用均匀的同位素标记的15 N，13 C标记的2′-脱氧寡核苷酸5′-dGpT和5′-dGpCpT获得的产率进行比较。分别从二寡核苷酸和三寡核苷酸衍生的d（G*pT*）和d（G*-T*）交联产物用作标准品，用于通过同位素稀释LC-MS/MS方法在选定的反应监测模式下鉴定小牛胸腺DNA中的类似病变。NIm和8-硝基-G是主要产物（每种约0.05%）和少量的8-氧代-G（G ~*pT ~*）和d（G ~*-T ~*）酶解产物结果表明，d（G*pT*）酶消化产物的形成只能由链内交联引起，而d（G*-T*）可由链间和链内交联产物产生。
Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•–and•NO2radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine–thymine product G*-T* with a covalent bond between the C8 (G*) and the thymine N3 (T*) atoms (CreanNucleic Acids Res. 2008, 36, 742−755). In this work, we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well-known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitro-G), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxo-G), and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and trioligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction monitoring mode. The NIm and 8-nitro-G are the major products formed (∼0.05% each), and lesser amounts of 8-oxo-G (∼0.02%) and d(G*pT*) and d(G*-T*) enzymatic digestion products (∼0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand and intrastrand cross-linked products.