Mammalian mitochondrial DNA is a closed circular duplex molecule of 16.5 kilobase pairs. The mechanism of mitochondrial DNA replication is controversial. The long-standing model of mammalian mitochondrial DNA replication entails continuous synthesis of the two strands of DNA.
More recently, it has been asserted that coupled leading and lagging-strand DNA synthesis is the major mechanism of mammalian mitochondrial DNA replication. Allegedly, the latter strand-coupled mechanism frequently involves extensive ribonucleotide incorporation on one strand, which gives rise to slow-moving replication fork arcs on neutral two dimensional agarose gels.
The identity of these arcs is disputed. It has been suggested that they might represent combined transcription/replication intermediates, rather than pure replication intermediates. In order to test this hypothesis and clarify the nature of molecular forms comprising the slow-moving replication fork arcs, I plan to isolate mitochondrial DNA molecules from cells and tissue s under conditions that retain RNA transcripts and analyse the material using both atomic force microscopy and two dimensional agarose gel electrophoresis.
This system also offers a means of studying the relationship between transcription and replication generally, including issues such as whether or not transcription and replication occur on the same molecule, and if so what is the effect of replication and transcription complexes colliding.
In a parallel study, I plan also to investigate ribonucleotide distribution in newly synthesised mitochondrial DNA.
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