A novel methodology for characterizing strand-break termini and damaged bases in plasmid DNA exposed to ionizing radiation

Abstract

We have developed a de novo methodology to characterize radiation damage in DNA. An enzyme system consisting of the 3′→5′ exonuclease snake venom phosphodiesterase (SVPD) and calf intestine alkaline phosphatase (CIAP) was used to examine the 3′ termini of strand-break sites. In this study, we hypothesized that the strand-break termini can be divided into two categories: CIAP-independent SVPD sites and CIAP-dependent SVPD sites. The former consists of strand-break termini that can be recognized directly and digested by SVPD without CIAP pretreatment, whereas the latter includes the termini that cannot be digested by SVPD without CIAP pretreatment. In addition, the apparent radiation–chemical yield ( G value) can be estimated using the level of intact 2′-deoxynucleotides produced during a 15-min incubation with SVPD. The G value for total strand breaks in fully dried DNA irradiated with 60Co γ-rays was estimated to be 0.1 μmol/J. Moreover, the G values of CIAP-dependent and CIAP-independent SVPD sites were estimated to be 0.078 and 0.024 μmol/J, respectively. These values suggest that 3′-phosphate termini are more likely to be produced than 3′ termini without phosphate. Furthermore, piperidine-treated irradiated plasmid DNA was also treated with the same enzyme system to examine the piperidine-labile sites. As a result of the treatment, the G value of the CIAP-dependent SVPD sites increased to 0.16 μmol/J, whereas no significant increase was seen in the G value of the CIAP-independent SVPD sites. This observation implies that most piperidine-labile damaged bases can be eliminated to form apurinic/apyrimidinic sites, which are completely removed by piperidine treatment to form 3′ phosphate termini, and that prompt CIAP-independent SVPD sites are piperidine resistant.