S 1 nuclease-specific nicking of mitochondrial DNA containing displacement loops

Abstract

Conditions are described in which the single strand-specific nuclease S 1 selectively nicks mitochondrial DNA containing displacement loops without nicking supercoiled mitochondrial DNA. Using these conditions, the percentage of molecules containing displacement loops can be easily and accurately determined. This method is superior to the traditional electron microscopic examination for assessing the frequency of displacement loop-containing molecules. In addition, this method permits the determination of the relative specific activities of displacement loop and nondisplacement loop-containing mitochondrial DNA after various radioactive labeling protocols. S 1 nuclease is shown to cleave the displaced strand of the displacement loop, to partially degrade the 7S-initiation strand, but not to cleave the parental template strand complementary to the 7S-initiation strand. The final product is a nicked circular molecule with at least two breaks localized within the displacement loop region in only one of the two parental strands.