Single-stranded regions in yeast mitochondrial DNA revealed by pulsed-field gel electrophoresis

The Klenow fragment of E. coli DNA polymerase together with pulsed-field gel electrophoresis (PFGE) have been used to investigate the presence of single-stranded DNA (ssDNA) regions in yeast (Torulopsis glabrata) DNA. Electrophoretic profiles of total DNA from Rho+ (wild type) and Rho0 (no mitochondrial DNA) strains demonstrate that this method mediates the incorporation of labelled dATP into mitochondrial DNA (mtDNA), but not into chromosomal DNA. The majority of ssDNA (> 62%) has been found associated with the electrophoretically inert component, localized on the top of PFGE gels. Treatment with single-stranded nucleases allows the resolution of this immobile fraction into fast migrating, linear molecules of a heterogeneous size. The possibility that single-stranded tracts and their recombinogenic properties are responsible for the trapping of DNA in pulsed-field gels is discussed.