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首页> 外文期刊>Biochemistry >DNA-Protein Cross-Linking via Guanine Oxidation: Dependence upon Protein and Photosensitizer.
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DNA-Protein Cross-Linking via Guanine Oxidation: Dependence upon Protein and Photosensitizer.

机译：通过鸟嘌呤氧化的DNA蛋白质交联：对蛋白质和光敏剂的依赖。

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DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.
机译：当鸟嘌呤在快速猝灭实验中经历1-电子氧化时，就会形成DNA-蛋白质交联，并在此检查活性氧，蛋白质和光敏剂的重要性。在这些实验中，通过与DNA结合的光敏剂的氧化淬灭而产生的强氧化剂产生了氧化的鸟嘌呤碱基，该鸟嘌呤碱基与蛋白质反应形成共价加合物。这些交联被热哌啶裂解，而不是反应性氧的结果，因为通过氯仿抽提法测定，羟基自由基清除剂（甘露醇）和氧都不会影响DNA-组蛋白交联的产率。交联产率取决于蛋白质，随着组蛋白>细胞色素c>牛血清白蛋白的降低而降低。产量不取决于细胞色素的氧化状态，这表明铁细胞色素c减少鸟嘌呤自由基不能有效地与交联竞争。光敏剂强烈影响交联产率，其以Ru（phen）（2）dppz（2+）[phen = 1,10-phenothroline;根据测得的氧化电位，dppz =二吡啶并吩嗪]> Ru（bpy）（3）（2+）[bpy = 2,2'-联吡啶]> cr啶橙>乙啶。聚（dG-dC）中乙二胺的长期瞬态吸收信号证实，鸟嘌呤氧化对该光敏剂无效。从（32）P标记的40-mer的聚丙烯酰胺测序凝胶中，所有这些光敏剂均显示在5'-GG-3'步骤的5'G优先损伤鸟嘌呤，与1-电子氧化一致。对乙啶的进一步检查表明，它可以在组蛋白和质粒DNA（pUC19）之间产生交联，产量取决于淬灭剂。总而言之，这些结果说明了快速淬灭技术的多功能性，该技术是通过鸟嘌呤氧化产生生理上相关的DNA-蛋白质加合物，并扩大这种交联反应的范围，使其包括可能仅与DNA短暂缔合的蛋白质的方法。

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