PHYSGENE · Dissecting a minimal genome: a physical investigation of DNA transactions in mitochondria

I propose to unravel parts of the mechanochemistry of two key genome transactions: replication and transcription using an array of single-molecule techniques. To this end, I propose to study the minimal genomic machinery of mitochondria. Mitochondria are organelles in eukaryotic cells that contain their own DNA (mtDNA). Significant understanding of the biochemistry of DNA transactions in mitochondria is obtained from the in vitro reconstitution of mitochondrial replication and transcription. However, elucidating the physics of these molecular mechanisms has only just started. A major challenge of the coming decades will be dissecting and quantifying biological systems to such an extent that it makes predictive modeling possible. Single-molecule tools play a major role in this development. Hence, I plan within the scope of this proposal, to combine optical manipulation with powerful fluorescent techniques. This combination of single-molecule manipulation and fluorescence has much more potential than has been explored, so far. With the development of such tools complex biological systems can not only be controlled and measured but also visualized at the same time. The human mitochondrial genetic machinery is an ideal system for such an approach, because replication and transcription can be re-created in vitro with only seven proteins. Hence, this proposal represents a unique opportunity to quantitatively dissect a genetic machine that is actually accessible by biophysical tools. Finally, about 1 in 5000 humans suffers from a disease caused by mutations of in the mtDNA. The research proposed here will permit direct observation of proteins in action. Normal and dysfunctional proteins can thus be compared, permitting insight in the mechanistic basis of a disorder.