Nucleotide assignment of alkali-sensitive sites in mouse mitochondrial DNA

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.