Extranuclear activities of genome maintenance protein complexes in stress response

The proposed work aims to define mechanisms how the mitochondrial single-stranded DNA binding protein (mtSSB) together with other factors maintain mitochondrial DNA (mtDNA) integ-rity, especially under conditions of stress. This research will fill critical gaps in our understanding of mtDNA metabolism, which is crucial for cellular energy production and overall mitochondrial function.

Aim 1: Mechanistic insights into the maintenance of mtDNA. We aim to define the roles of mtSSB acting with DNA repair factors during mtDNA metabolism, particularly in response to DNA breaks and other types of DNA damage, as well as secondary DNA structures such as G-quadruplexes (G4) and R-loops. The experiments will involve in vitro biochemical and cellular experiments to define the roles of these interactions in the overall mitochondrial function.

Aim 2: Unbiased identification of novel factors and interactions in mtDNA maintenance. Besides candidate-based approaches, our research will also employ high-throughput screens to identify novel factors involved in mtDNA metabolism as well as their associated physiological interaction networks.

Aim 3: Definition of phase-separated mtDNA metabolic compartments. We aim to under-stand the role of liquid-liquid phase separation (LLPS) in the compartmentalization and regulation of mtDNA metabolic processes. Using advanced biophysical methods such as fluorescence mi-croscopy and mass photometry, the team will study the propensity of mtDNA metabolic proteins to undergo phase separation and form functional compartments. This aim also includes the exam-ination of how redox conditions affect these processes and the dynamics of protein-nucleic acid interactions within phase-separated condensates.

Aim 4: The role of protein transport in response to mtDNA stress. Knowledge about mtDNA maintenance mechanisms will be placed into a broader cellular context by determining how pro-tein transport mechanisms influence mtDNA stability and cellular responses to mitochondrial stress.

In summary, this proposal aims to make significant contributions to the field of mitochondrial biology by defining new mechanisms of mtDNA maintenance, centered around the mtSSB protein, and exploring innovative regulatory pathways involving phase-separated membraneless com-partments as well as the trafficking of mtDNA metabolic factors between cytosolic and mitochon-drial compartments. The work is multidisciplinary, leveraging the expertise of three leading re-search groups with complementary skills in biochemistry, molecular biology, and mitochondrial physiology. The findings are expected to have broad implications for our understanding of mito-chondrial function in health and disease.