Kurbanyan Kristina, Nguyen Kim L, To Phuong, Rivas Eunice V, Lueras Alexis M K, Kosinski Cynthia, Steryo Mary, González Arcelia, Mah Daisy Ann, Stemp Eric D A
Department of Physical Sciences and Mathematics, Mount St. Mary's College, Los Angeles, California 90049, USA.
Biochemistry. 2003 Sep 2;42(34):10269-81. doi: 10.1021/bi020713p.
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.
在快速淬灭实验中，当鸟嘌呤发生单电子氧化时会形成DNA-蛋白质交联，本文研究了活性氧、蛋白质和光敏剂的重要性。在这些实验中，与DNA结合的光敏剂通过氧化淬灭产生的强氧化剂会生成氧化鸟嘌呤碱基，该碱基与蛋白质反应形成共价加合物。这些交联可被热哌啶裂解，且不是活性氧的作用结果，因为通过氯仿萃取测定法可知，羟基自由基清除剂（甘露醇）和氧气均不影响DNA-组蛋白交联的产率。交联产率取决于蛋白质，其降低顺序为组蛋白>细胞色素c>牛血清白蛋白。产率不取决于细胞色素的氧化态，这表明亚铁细胞色素c对鸟嘌呤自由基的还原与交联反应之间没有有效的竞争。光敏剂对交联产率有很大影响，根据测得的氧化电位，其降低顺序为Ru(phen)(2)dppz(2+) [phen = 1,10-菲咯啉；dppz = 二吡啶并菲嗪]> Ru(bpy)(3)(2+) [bpy = 2,2'-联吡啶]>吖啶橙>溴化乙锭。聚（dG-dC）中溴化乙锭二价阳离子的长寿命瞬态吸收信号证实，该光敏剂对鸟嘌呤的氧化效率较低。从一个(32)P标记的40聚体的聚丙烯酰胺测序凝胶中可以看出，所有这些光敏剂均优先在5'-GG-3'序列的5'端鸟嘌呤处损伤鸟嘌呤，这与单电子氧化一致。对溴化乙锭的进一步研究表明，它可在组蛋白和质粒DNA（pUC19）之间产生交联，且产率取决于猝灭剂。总之，这些结果说明了快速淬灭技术作为一种通过鸟嘌呤氧化生成生理相关DNA-蛋白质加合物的方法的多功能性，并将此类交联反应的范围扩展到包括可能仅与DNA瞬时结合的蛋白质。
