Novel Lines of Evidence for the Asymmetric Strand Displacement Model of Mitochondrial DNA Replication.

The mitochondrial genome, which consists of 16,569 bp of DNA with a cytosine-rich light (L) strand and a heavy (H) strand, exists as a multicopy closed circular genome within the mitochondrial matrix. The machinery for replication of the mammalian mitochondrial genome is distinct from that for replication of the nuclear genome. Three models have been proposed for mitochondrial DNA (mtDNA) replication, and one of the key differences among them is whether extensive single- C) stranded regions exist on the H strand. Here, three different methods that can detect single-stranded DNA (ssDNA) are utilized to identify the presence, location, and abundance of ssDNA on mtDNA. Importantly, none of these newly described methods involve the complication of prior mtDNA fractionation. The H strand was found to have extensive single-stranded regions with a profile consistent with the strand displacement model of mtDNA replication, whereas single strandedness was predominantly absent on the L strand. These findings are consistent with the in vivo oc-cupancy of mitochondria! single-stranded DNA binding protein reported previously O and provide strong new qualitative and quantitative evidence for the asymmetric strand displacement model of mtDNA replication.