Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid

Abstract

Supercoiled rat liver mitochondrial DNA is relaxed by treatment with RNase A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to RNase H and A, but only 35% of the supercoiled population is sensitive to RNase T1. Removal of the ribonucleotides with calf thymus RNase H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the EM, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to 1 another. The average number of ribonucleotide sites per molecule was estimated to be between 8-13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.