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, how ever, has not been considered, even though reversible DNA-protein interacti ons are key to gene expression and to DNA replication and repair. In the cu rrent studies, we show that hypochlorous acid (HOCl), generated by the myel operoxidase-hydrogen peroxide-chloride system of phagocytes, cross-links si ngle-stranded DNA-binding protein (SSB) to single-stranded oligonucleotides . Exposure of SSB and a homopolymer of radiolabeled thymidine (dT(40)) to H OCl resulted in the formation of a radiolabeled band with slower mobility t han the free oligonucleotide, as determined by denaturing polyacrylamide ge l electrophoresis. This radiolabeled band did not appear if the reaction mi xture was treated with protease or nuclease, indicating that it represents a covalent complex of DNA and protein. Oligonucleotides of adenosine and cy tidine behaved similarly to the thymidine oligonucleotide, demonstrating th at they are also capable of participating in the cross-linking reaction. Th e covalent complex of radiolabeled dT40 and SSB was also generated by chlor amines and the complete myeloperoxidase-hydrogen peroxide-chloride system. The enzymatic reaction required each component of the system and was inhibi ted by heme poisons and chloride-free conditions, implicating myeloperoxida se and HOCl. DNA-protein cross-links were generated in Escherichia coli exp osed to HOCl, suggesting that double-stranded DNA is also a target for the reaction. These results indicate that long-lived chloramines and HOCl gener ated by myeloperoxidase can generate covalent DNA-protein cross-links that may contribute to the mutagenic and cytotoxic effects of phagocytes on micr obial pathogens and host tissue.