DIFFERENTIAL-EFFECTS OF DNA SUPERCOILING ON RADICAL-MEDIATED DNA STRAND BREAKS

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is a ffected by the alterations in DNA structure and dynamics that accompan y superhelical tension, we have investigated he effect of torsional te nsion on DNA damage produced by five oxidizing agents: gamma-radiation , peroxynitrite, Fe2+/EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positi vely supercoiled plasmid DNA prepared by a recently developed techniqu e, we compared the quantity of strand breaks produced by the five agen ts in negatively and positively supercoiled pUC19. It was observed tha t strand breaks produced by gamma-radiation, peroxynitrite, and Fe(2+) EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly re active intermediates (e.g., hydroxyl radical), but do not interact dir ectly with DNA, will be relatively insensitive to the changes in DNA s tructure and dynamics caused by superhelical tension, In the case of F e2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sen sitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O 2 in the positively supercoiled substrate occurred at lower Cu concent rations than in negatively supercoiled DNA, Furthermore a sigmoidal Cu 2+/H2O2 damage response was observed in the negatively supercoiled sub strate but not in positively supercoiled DNA, The results with Cu2+ su ggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigat ion into the role of supercoiling in base damage.