In vitro protection against radiation damage to template activity in DNA synthesis.

Abstract

SummaryIn vitro protection against radiation-induced functional changes in DNA was studied by measuring DNA template activity in enzymatic DNA synthesis. Exposures up to 28 kR inhibited the priming activity of salmon sperm DNA with E. coli DNA-polymerase, reducing the incorporation of all four deoxynucleotides and altering the incorporation kinetics. The radiation protective agent bis(2-guanidoethyl) disulphide (GED) protected against these changes. GED-protected-DNA exposed to 7 and 14 kR actually showed enhanced template activity, incorporating 20–50 per cent more dGMP than the unirradiated control. Partial denaturation of DNA by dissolving in distilled water and complete denaturation by heat, before irradiation, increased the radiosensitivity of DNA. Heat-denaturation of unirradiated DNA did not affect its template activity, but heat-denaturation of DNA previously irradiated in double-stranded form reduced its template activity below that of irradiated undenatured DNA. Denaturation may release inactive fragments produced by radiation-induced single-strand breaks. Radiation alteration of the template resulted in different effects on the incorporation of the different deoxynucleotides, indicating miscoding. GED protected against this effect by tending to maintain the normal deoxynucleotide ratio. CsCl density-gradient equilibrium centrifugation of irradiated DNA showed a shift to higher density, and this was not protected against by GED. This may represent localized denaturation (and cross-linking) and may explain the enhanced DNA template activity, observed under certain conditions.