Determination of G-values for Single and Double Strand Break Induction in Plasmid DNA Using Agarose Gel Electrophoresis and a Curve-fitting Procedure

Summary

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments.

DNA (30–800 μg/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D₃₇ values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots.

The following yields were determined: G_ssb 3·4 × 10E–8 mol J⁻¹, and G_dsb 3·3 × 10E–10 mol J⁻¹. G_dsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.