An improved approach to the analysis of plasmid DNA breakage by decay of DNA-associated auger emitters.

PURPOSE: To assess DNA breakage by DNA-associated decay of the Auger electron emitter 125I, using an improved analytical approach.

MATERIAL AND METHODS: Breakage of pBR322 plasmid DNA following incubation with DNA-binding ligand Hoechst 33258 labelled with 125I was studied by measuring the conversion of native supercoiled to relaxed and linear forms. The analytical approach that was developed considers two distinct sources of breakage: DNA damage at the site of DNA-associated Auger decay (local effect), and DNA damage from the radiation field created by all decay events in the incubation volume (external breakage). In addition, to enable application of Poisson statistics to local breakage events, relaxation events and linearisation events are considered rather than single-stranded and double-stranded breaks.

RESULTS: The results indicated that a decay event of DNA-associated 125I induced relaxation and linearisation of the plasmid molecule with probabilities of 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. Addition of a radical scavenger DMSO increases the probability of relaxation to 0.15 +/- 0.02 and reduces the probability of linearisation to 0.65 +/- 0.05.

CONCLUSIONS: Accurate evaluation of the local effect in plasmid breakage requires consideration of external breakage, the contribution of which depends on experimental conditions. The highly efficient production of linearisation events by (125)I-decay restricts the detection of relaxation events by the plasmid assay.