Periodic Reporting for period 3 - MitoRepairosome (Dissecting the mechanism of DNA repair in human mitochondria)
Reporting period: 2023-02-01 to 2024-07-31
Human cells contain hundreds of mitochondria, each housing multiple DNA molecules that encode essential components of oxidative phosphorylation (OXPHOS). While OXPHOS plays a critical role in providing cellular energy in the form of ATP, it generates reactive oxygen species (ROS) that can potentially damage mitochondrial DNA (mtDNA). Mutations in mtDNA can lead to mitochondrial disorders characterized by a wide range of clinical manifestations and have been associated with various conditions, including cancer, premature aging, cardiovascular disorders, skeletal muscle issues, and neurological disorders.
To counteract damage from ROS, mitochondria developed a robust DNA repair mechanism known as the DNA base excision repair (BER) pathway which serves as a primary defense mechanism against oxidative damage in human mitochondria. Strategically positioned in vicinity of the inner mitochondrial membrane, essential BER enzymes including EXOG, APE1, PolγAB, and Lig3 collaborate to create a large molecular complex known as the 'mitochondrial repairosome' which is capable of carrying out complete DNA repair.
The goal of this research project is to study the structure, mechanism and function of human mitochondrial repairosome. We will utilize a combination of structural biology (x-ray crystallography and electron microscopy), biochemistry and biophysics and cell biology by investigating the following aims:
Aim 1: Reconstitute a scaffold of mitochondrial repairosome on the membrane
Aim 2: Investigate functional synergy between components of mitochondrial repairosome
Aim 3: Investigate the structures of mitochondrial repairosome
To counteract damage from ROS, mitochondria developed a robust DNA repair mechanism known as the DNA base excision repair (BER) pathway which serves as a primary defense mechanism against oxidative damage in human mitochondria. Strategically positioned in vicinity of the inner mitochondrial membrane, essential BER enzymes including EXOG, APE1, PolγAB, and Lig3 collaborate to create a large molecular complex known as the 'mitochondrial repairosome' which is capable of carrying out complete DNA repair.
The goal of this research project is to study the structure, mechanism and function of human mitochondrial repairosome. We will utilize a combination of structural biology (x-ray crystallography and electron microscopy), biochemistry and biophysics and cell biology by investigating the following aims:
Aim 1: Reconstitute a scaffold of mitochondrial repairosome on the membrane
Aim 2: Investigate functional synergy between components of mitochondrial repairosome
Aim 3: Investigate the structures of mitochondrial repairosome
The team, composed of two postdoctoral scientists, a research technician, and me, has made significant progress in all three Aims. First, we have developed tools that enable the reconstitution of full-length EXOG in membrane mimetic systems, facilitating the study of its interactions with the membrane. A manuscript detailing these findings is currently in preparation. Second, we have investigated the formation of binary complexes between components of the mitochondrial repairosome and are currently exploring the organization of the repairosome in the presence of DNA. Third, we used biochemical and biophysical methods to examine the functional synergy between mitochondrial repairosome components and the impact of oxidative damage on their activity. We have shown that that the activity and interactions between mitochondrial BER enzymes are significantly modulated by ROS. The first manuscript detailing these discoveries is currently being prepared. Additionally, we are engaged in the structural characterization of several sub-repairosome complexes using X-ray crystallography and single-particle cryo-EM.
The human mitochondrial repairosome is a highly complex molecular machinery composed of several enzymes. Its flexible nature poses a significant challenge for both biochemical and structural biology studies. The techniques developed in this project will provide insights into how the human mitochondrial repairosome executes DNA repair transactions. These new insights will not only enhance our understanding of a fundamental aspect of mitochondrial DNA metabolism but also contribute to our understanding of the impact of oxidative damage on the effectiveness of DNA repair pathways.