Quiñones Jason L, Thapar Upasna, Yu Kefei, Fang Qingming, Sobol Robert William, Demple Bruce
Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY 11794;
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824;
Proc Natl Acad Sci U S A. 2015 Jul 14;112(28):8602-7. doi: 10.1073/pnas.1501101112. Epub 2015 Jun 29.
Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them.
自由基对DNA脱氧核糖C1'位置的攻击会产生氧化脱碱基（AP）位点2-脱氧核糖内酯（dL）。遇到dL时，诸如DNA聚合酶β（Polβ）等AP裂解酶在体外进行DNA修复尝试时会形成终末共价中间体。然而，导致此类DNA-蛋白质交联（DPC）在体内形成的条件及其对细胞功能的影响仍不清楚。我们采用免疫斑点印迹法在体内检测氧化性Polβ-DPC。用在DNA中产生dL的遗传毒性氧化剂处理哺乳动物细胞会导致体内形成Polβ-DPC。以剂量依赖方式，在用抗肿瘤药物替拉扎明（TPZ；在1% O₂ 下比在21% O₂ 下形成更多的Polβ-DPC）处理的MDA-MB-231人细胞中检测到Polβ-DPC，在用“化学核酸酶”1,10-邻菲罗啉铜（Cu(OP)₂）处理时检测到的更明显。用TPZ或Cu(OP)₂ 处理的小鼠胚胎成纤维细胞也会产生Polβ-DPC。非氧化性试剂不会产生Polβ-DPC。体内交联显然是碱基切除DNA修复途径的结果：氧化性Polβ-DPC依赖于产生Polβ裂解酶底物的Ape1 AP核酸内切酶，并且它们需要Polβ裂解酶活性位点中的必需赖氨酸-72。氧化性Polβ-DPC在人和小鼠细胞中的半衰期出人意料地短（约30分钟），其去除依赖于蛋白酶体。在Cu(OP)₂ 处理下蛋白酶体抑制对表达野生型Polβ的细胞比对照裂解酶缺陷型细胞的细胞毒性明显更大。该观察结果强调了氧化性Polβ-DPC的遗传毒性潜力以及修复它们的生物学压力。
