BIOLOGICAL CONSEQUENCES OF DNA-DAMAGE INTRODUCED IN BACTERIOPHAGE-PM2DNA BY HYDROGEN PEROXIDE-MEDIATED FREE-RADICAL REACTIONS

In order to study the biological consequences of DNA damage induced by H2O2-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H2O2, Fe3+-citrate and ascorbate either alone or in combina tion, Induction of DNA lesions was determined as well as the biologica l activity of the phage DNA, Exposure to H2O2 alone resulted in max, 0 .2 single-strand breaks per molecule; in the presence of Fe3+-citrate, the yield was similar to 4-fold higher. Under both conditions no doub le-strand breaks could be detected and the biological activity was not diminished, This indicates that low levels of single-strand breaks as generated by H2O2/Fe3+-citrate do not inactivate PM2 DNA, Exposure to ascorbate in the presence Fe3+-citrate resulted in extensive inductio n of single-strand breaks, However, at ascorbate concentration where s imilar to 3 single-strand breaks per molecule were induced, again no d ouble-strand breaks could be detected and the biological activity of t he DNA was not diminished, At 5 mM ascorbate, single-strand breaks wer e above the detection limit, Under these conditions, 0.02 double-stran d breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation wa s calculated to be similar to 3%. When PM2 DNA was exposed to H2O2 in the presence of ascorbate/Fe3+-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand bre aks and biological inactivation, suggesting that one or more reactive species sensitive to H2O2 play a critical role, The (OH)-O-. scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA , which may indicate that other reactive species than (OH)-O-. are inv olved, Our data suggest that other reactive species than (OH)-O-., suc h as the ferryl ion, are involved in H2O2-mediated DNA damage inductio n and biological inactivation.