Kulcharyk P A, Heinecke J W
Departments of Medicine, Biochemistry and Biophysics, and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2001 Mar 27;40(12):3648-56. doi: 10.1021/bi001962l.
Phagocytic oxidants have been implicated in tissue injury and oncogenesis, and their pathophysiological role in modifying nucleobases and amino acids has been widely explored. Their ability to cross-link proteins and DNA, however, has not been considered, even though reversible DNA-protein interactions are key to gene expression and to DNA replication and repair. In the current studies, we show that hypochlorous acid (HOCl), generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes, cross-links single-stranded DNA-binding protein (SSB) to single-stranded oligonucleotides. Exposure of SSB and a homopolymer of radiolabeled thymidine (dT(40)) to HOCl resulted in the formation of a radiolabeled band with slower mobility than the free oligonucleotide, as determined by denaturing polyacrylamide gel electrophoresis. This radiolabeled band did not appear if the reaction mixture was treated with protease or nuclease, indicating that it represents a covalent complex of DNA and protein. Oligonucleotides of adenosine and cytidine behaved similarly to the thymidine oligonucleotide, demonstrating that they are also capable of participating in the cross-linking reaction. The covalent complex of radiolabeled dT(40) and SSB was also generated by chloramines and the complete myeloperoxidase-hydrogen peroxide-chloride system. The enzymatic reaction required each component of the system and was inhibited by heme poisons and chloride-free conditions, implicating myeloperoxidase and HOCl. DNA-protein cross-links were generated in Escherichia coli exposed to HOCl, suggesting that double-stranded DNA is also a target for the reaction. These results indicate that long-lived chloramines and HOCl generated by myeloperoxidase can generate covalent DNA-protein cross-links that may contribute to the mutagenic and cytotoxic effects of phagocytes on microbial pathogens and host tissue.
吞噬性氧化剂与组织损伤和肿瘤发生有关，并且它们在修饰核碱基和氨基酸方面的病理生理作用已得到广泛研究。然而，尽管可逆的DNA-蛋白质相互作用是基因表达以及DNA复制和修复的关键，但它们交联蛋白质和DNA的能力尚未得到考虑。在当前的研究中，我们发现吞噬细胞的髓过氧化物酶-过氧化氢-氯化物系统产生的次氯酸（HOCl）能使单链DNA结合蛋白（SSB）与单链寡核苷酸交联。通过变性聚丙烯酰胺凝胶电泳测定，将SSB和放射性标记的胸腺嘧啶同聚物（dT(40)）暴露于HOCl会导致形成一条迁移率比游离寡核苷酸慢的放射性标记条带。如果用蛋白酶或核酸酶处理反应混合物，则不会出现这条放射性标记条带，这表明它代表DNA和蛋白质的共价复合物。腺苷和胞苷的寡核苷酸表现与胸腺嘧啶寡核苷酸相似，表明它们也能够参与交联反应。放射性标记的dT(40)与SSB的共价复合物也可由氯胺和完整的髓过氧化物酶-过氧化氢-氯化物系统产生。酶促反应需要该系统的每个组分，并且受到血红素毒物和无氯化物条件的抑制，这表明髓过氧化物酶和HOCl参与其中。暴露于HOCl的大肠杆菌中产生了DNA-蛋白质交联，这表明双链DNA也是该反应的靶点。这些结果表明，髓过氧化物酶产生的长寿命氯胺和HOCl可产生共价DNA-蛋白质交联，这可能导致吞噬细胞对微生物病原体和宿主组织产生诱变和细胞毒性作用。
