Studies on the organelle-specific functions of human Type 1A topoisomerase TOP3A

Mitochondrial DNA depletion syndromes are a group of clinically heterogeneous genetic diseases that affect patients in infancy and childhood. Hitherto nine genes have been identified to be associated to these diseases, however, there are a considerable number of verified cases, where the mutated gene responsible for the disease has not been identified. The basic science research project proposed in this application will considerably enhance our understanding of the molecular mechanisms responsible for mitochondrial DNA depletion syndromes, and open up further avenues for the treatment of its sufferers.

In most living organisms biomaterial that builds the body of cells is coded in the DNA. This coded information is read in a process termed RNA transcription. Before the division of cells, in order to maintain the equal distribution of DNA content between the daughter cells, the DNA has to be duplicated; this process is termed DNA replication. During these rather complicated processes, the DNA has to open up to give access to enzymes that function in DNA metabolism. Separation of the two strands of DNA is catalysed by helicase enzymes. During the unwinding of the DNA double helix by the helicase enzymes, torsional stress is generated, which is relieved by another set of enzymes called topoisomerases. Topoisomerases are also capable of tidying up tangled stretches of DNA.

The powerplant of cells, the mitochondria, also contain DNA, but - curiously enough - this DNA is circular like DNA of most bacterial cells, and unlike the linear chromosomal DNA that can be found in the nucleus of eukaryotes. Mitochondrial DNA is more susceptible to damage than nuclear DNA because of the metabolic processes that take place in the mitochondria.

One of the enzymes that guard the integrity of nuclear as well as mitochondrial DNA is DNA topoisomerase III alpha (TOP3A). The complete lack of this enzyme in mice causes lethality during early embryonic development, while its reduced levels in cell culture cause the accelerated ageing of cells. Though the biochemical activities of TOP3A have been well characterised, its exact function is largely unknown, and even less is known about its role in maintaining the integrity of the mitochondrial genome.

In my preliminary work I have set up a system that allows the depletion of TOP3A from the cells, while specific expression and function can be maintained in either the nuclei or in the mitochondria, exclusively. This system permits the analysis of nuclear and mitochondrial functions of TOP3A independently of each other to reveal why the ageing process is accelerated in cells devoid of TOP3A, and whether it can be attributed to a function of the enzyme specific to the nuclei or the mitochondria. More specifically, we will investigate how the replication of mitochondrial DNA, and the metabolic activity of mitochondria are affected in cells that lack mitochondrial TOP3A function. We will study how suppression of nuclear TOP3A functions affects nuclear DNA metabolism, chromosome segregation and cell divisions. We will also identify and characterise protein partners of TOP3A that assist its organelle-specific functions.

Despite the recent advances in the biochemistry of TOP3A, it is not clear what processes it participates in the nucleus and what its role is in the maintenance of mitochondrial DNA (mtDNA). The phenotypic consequences of TOP3A depletion are also rather complex and difficult to attribute to either the nuclear or mitochondrial role of the enzyme.

In this proposal we will dissect the essential cellular role of TOP3A in maintaining the integrity of nuclear and mitochondrial DNA. In my preliminary work I have set up a system that permits the selective expression of either the nuclear or the mitochondrial form of TOP3A in the background of its systemic RNAi-mediated depletion. We will use this system to verify the phenotypic consequences of TOP3A depletion and to attribute each of the phenotypes to either the mitochondrial or the nuclear function of the enzyme. These experiments will be based on microscopic observations following staining with specific antibodies or the senescence marker beta-galactosidase.

We will study how elimination of TOP3A from the mitochondria affects mtDNA replication and mitochondrial metabolic activity. We will use two-dimensional agarose gel electrophoresis (N2D-AGE) to visualise replication intermediates and other DNA structures and quantitative real-time PCR to measure mtDNA copy number. Metabolic activity of mitochondria will be monitored with measuring ATP and ROS concentrations and mitochondrial membrane potential.

We will map the protein interaction network of TOP3A both in the mitochondria and in the nucleus. GFP tagged mitochondrial or nuclear forms of TOP3A will be expressed in cells and the tagged protein will be specifically pulled down using the GFP-nanotrap technology. Proteins specifically bound to TOP3A will be identified by mass spectrometry. The interaction will be verified with in vitro (co-immunoprecipitation, far-Western), and microscopy-based cellular techniques (FRET, bimolecular fluorescent complementation).

Loss of mitochondrial DNA or damage to mtDNA leads to disturbances in the metabolic activity of mitochondria, which has been shown to be associated with the ageing process and mtDNA depletion syndromes (MDS). This project will deliver key information about the role of DNA topoisomerase IIIalpha in mtDNA maintenance and its effect on the metabolic activity of mitochondria. With its deliverables it will open up new avenues for the benefit of the research community, for training and public engagement, end for exploitation of basic research findings in healthcare and commercial applications.

Our findings will be published in "open access" publications to ensure to a wide research community. Research findings will be communicated in international and national conferences before publication to receive helpful feedback and criticism. We will seek collaborative connections initially to share new and improved techniques ideas, preliminary data and reagents, then set up new collaborative projects in joint grant proposals.

It has been my ambition to maintain a laboratory portal for many years, and with the infrastructure provided by the University of Lincoln it will be set up maintained and updated for years to come. We will use this service to communicate our findings to expert scientists and to share "raw" experimental data, but also to communicate our findings to the wider audience to emphasise the importance of our research. In addition to public outreach through the website, we will maintain already existing links and set up new ones with Lincolnshire schools to promote interest in our work, and organise demonstrations and lectures to actively involve young people in science.

We are aiming to hire an experienced postdoctoral researcher, in return we will support her/his career development and aspirations to e.g. become an independent researcher or join a commercial enterprise in biotechnology. The PDRA will be actively involved in networking with academic and clinical researchers, and with potential industrial partners. We will recruit PhD and MSc students to complement research not only manually but with ideas as well, and to provide valuable technical and theoretical training.

We will seek collaborations with clinicians who provide treatment and care for mitochondrial DNA depletion syndrome sufferers, to set up collaborations for follow-up studies to test the involvement of Topo IIIalpha in the aetiology of these diseases. This will also open up avenues to identify "druggable" targets with potential commercial partners.

In our review process a high priority will be given to assess if the impact objectives are met and to identify new avenues of impact delivery. Person responsible for impact management will be the PI.