Hypochlorous acid produced by the myeloperoxidase system of human phagocytes induces covalent cross-links between DNA and protein.
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.
Kulcharyk
P A
PA
Departments of Medicine, Biochemistry and Biophysics, and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Heinecke
J W
JW
D002700:Chloramines / Q000737:chemistry; D002712:Chlorides / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003853:Deoxyribonucleosides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology* / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013997:Time Factors
D002700:Chloramines; D002712:Chlorides; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D003853:Deoxyribonucleosides; D007364:Intercalating Agents; D016877:Oxidants; D011108:Polymers; D012710:Serum Albumin, Bovine; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; C030816:chloramine