Single-strand Breaks can Lead to Complex Configurations of Plasmid DNA in Vitro

Summary

DNA species which migrate extremely slowly in agarose gel electrophoresis may be formed from plasmid DNA containing radiation-induced single-strand breaks (ssbs). Postirradiation heat treatment in low ionic strength buffer and subsequent incubation with Mg²⁺ strongly enhanced the formation of these species. Electron micrographs taken after such treatment show numerous complex configurations containing DNA material from several plasmid molecules. Less extreme formation of slowly migrating DNA occurred without postirradiation heat treatment or Mg²⁺ incubation when the DNA was co-precipitated with calcium phosphate in a physiologically balanced buffer and incubated under conditions used for DNA-mediated gene transfer. The data suggest that homologous pairing between single-stranded regions formed in relation to ssbs may contribute to cohesion between different molecules. The significance of the cohesion process for gene transfer experiments and cellular radiation effects is discussed.