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

Final Report Summary - PHYSGENE (Dissecting a minimal genome: a physical investigation of DNA transactions in mitochondria)

One of the major challenges of the coming decades will be dissecting and quantifying biological systems to such an extent that it makes predictive modelling of these systems possible. Single-molecule tools such as Optical/Magnetic Tweezers, Fluorescent Microscopy and Atomic Force Microscopy are in constant development and as a result increasingly complex biological systems can be studied. In this project, we are succeeding to bring this dream a significant step closer, by combining optical manipulation with powerful fluorescent techniques, such as confocal and super-resolution technology. The new instrument that we have developed represents the state of the art and is able to make real-time movies of many proteins binding, handling and moving about on DNA. Most significantly the microscope can do this at high protein concentrations on and around the DNA which is exactly like the situation inside cells. This combination of single-molecule manipulation and fluorescence has an enormous potential for the quantification of biological systems. Moreover, the instrument is very flexible in how it can be used and therefore it permits the study of any kind of DNA-proteins.

Moreover, the ERC project illustrated nicely serendipity in action. While working on the technology of super resolution with tweezers we started to think about how to speed up some measurements by doing many single molecule experiments in parallel. When I visited a lab in Innsbruck working with sound waves in order to manipulate small water animals, I realized that sound waves might also be suitable to manipulate molecules. Sound waves have long wavelengths with respect to molecules and are easy to generate over a large surface area, thus creating the potential for doing measurements in parallel. Within about one year, we worked out an instrumental configuration that would allow us to do this on many DNA molecules. This work appeared last year in Nature Methods and got a lot of attention from scientist as well as the life-science industry. As a result we are now selling the technology world-wide through, our ERC PoC funded startup, just one year after the publication of the technology.
One of our major goals of the development of our instruments was to unravel the genetic machinery. Many genetic processes can be explored using our approach of filming DNA-protein interactions. The reason for that is that from quite a number of organisms replication, transcription and DNA organization can be re-created in a test-tube. As a result, it is possible to generate these mechanisms in our microscope, hence creating a unique opportunity to quantitatively dissect complex genetic machines. Moreover, malfunction of such genetic machinery is implicated in quite a number of diseases. Therefore, studying normal and dysfunctional genetic functioning will permit insight in the mechanistic basis of genetic disorders.
Within this project we have succeed in revealing many details about the mechanism of DNA organization. In particular, we found that the DNA gets compacted by the protein TFAM, the most abundant protein DNA-binding protein inside human mitochondria. How it accomplishes this compaction is surprising; it forms filaments on the DNA and within these filaments it generates small melting bubbles. Melted DNA is very flexible and acts as hinge. It is the introduction of many of those hinges that permits the strong compaction of the DNA. We also demonstrated that compacted DNA is not accessible for replication and transcription and therefore TFAM concentrations can be used as a global regulator for the access of mitochondrial DNA. We published 3 manuscripts on TFAM and a 4th one on the impact of post-translational modifications in on its way. We have also successfully reconstituted many individual parts of the T7 replisome as well as many of the components together. Several manuscripts are pending at the moment.