Biological consequences of DNA damage introduced in bacteriophage PM2 DNA 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 combination. Induction of DNA lesions was determined as well as the biological 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 ~4-fold higher. Under both conditions no double-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 induction of single-strand breaks. However, at ascorbate concentration where ~3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished. At 5 mM ascorbate, single-strand breaks were above the detection limit. Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation was calculated to be ~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 breaks and biological inactivation, suggesting that one or more reactive species sensitive to H₂O₂ play a critical role. The .OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than .OH are involved. Our data suggest that other reactive species than .OH, such as the ferryl ion, are involved in H₂O₂-mediated DNA damage induction and biological inactivation.