Biochemical Evaluation of Human DNA-Lysine Photoadduct Treated with Peroxynitrite

ABSTRACT

Peroxynitrite is a reactive oxidant produced from nitric oxide (.NO) and superoxide anion (O₂^.−). It is produced by the body in response to environmental toxins, stress, ultraviolet light, ischemia/reperfusion, inflammation, etc. In vivo, peroxynitrite is formed in macrophages, endothelial cells, platelets, leukocytes, and neurons. It reacts with a variety of biomolecules including proteins, lipids, and DNA. We have investigated the photochemical addition of lysine to native DNA in view of its potential importance in the photo-cross-linking of histones to DNA in chromatin. Lysine-and arginine-rich histone H1 in nucleosome on modification by physical, chemical, or environmental agents forms histone-DNA adducts. We have characterized the photoadducts by absorption, fluorescence, and chromatographic methods. The UV absorption spectra of the DNA-lysine photoadduct showed hyperchromism, indicating structural distortions in DNA either due to single-strand breaks or opening of the double helix at the site of lysine conjugation. On peroxynitrite treatment, the melting temperature (T_m) of the DNA-lysine adduct increased by 15°C compared to the native DNA-lysine adduct. A decrease in the fluorescence intensity of the DNA-lysine photoadduct with respect to the modified adduct was observed. The gel filtration profile of the peroxynitrite-modified adduct was also found to be different from that of the native DNA and DNA-lysine photoadduct. Hence, the peroxynitrite-modified photoadduct may have important implications in toxicology, mutagenesis, and carcinogenesis.

KEYWORDS:

• Human DNA
• Lysine
• Peroxynitrite
• Photoadduct