Project No. 2302
Dr Robert Baldock – University of Portsmouth
Prof Sarah Newbury – University of Sussex, Brighton and Sussex Medical School
Prof Garry Scarlett – University of Portsmouth
Maintaining the integrity of the mitochondrial genome (mtDNA) is central to normal cellular function, and enabling cells to tolerate stress.
An evolving picture of mtDNA repair mechanisms is revealing how cells repair and tolerate damage. Importantly, nuclear DNA repair insights show RNA to be integral role in maintaining nuclear genomic integrity. Crucially, the balance between DNA repair and targeted degradation of damaged mtDNA copies is likely to be an important mechanistic step in the pathogenesis mitochondrial-associated diseases. Despite this, the mechanism of degradation as well as the factors that control the selection and degradation of damaged mtDNA remain unknown.
This project aims to elucidate the role of nucleases in mitochondrial DNA degradation and repair. Using a targeted informatics screen to identify candidate nucleases, we will combine mammalian cell culture with the versatility of Drosophila genetics to selectively downregulate candidate nucleases and study their impact on mtDNA integrity, stability, and overall mitochondrial function.
This project will involve studying the mitochondrial localisation and interaction of specific nucleases with DNA/RNA using cellular fractionation, immunoblotting and nucleic acid-protein proximity ligation assays. In nuclease-depleted cells, mtDNA copy number variation will be determined by qPCR in response to different mitotoxic stresses. Finally, mitochondrial oxygen consumption, reactive oxygen species production and mitochondrial membrane potential will be characterised using high-throughput analysers to evaluate overall impact on mitochondrial function.
This project will lead to a new understanding of the mitochondrial-associated pathogenesis by investigating the quality control mechanism(s) that degrade damaged mtDNA.