Year: 2021
Type: dissertation
Abstract: Mitochondria are found in eukaryotic cells, and they are responsible for many cellular functions including energy metabolism, apoptosis, calcium handling, and synthesis pathways.Mitochondria has its own genome though majority of mitochondrial proteins are encoded in nuclear DNA (nDNA).Mitochondrial DNA (mtDNA) is more prone to damage than nDNA.Damage to mtDNA can result in several mitochondrial myopathies; therefore, mtDNA integrity is crucial for normal cell function.MRE11 is known as a nuclease; it forms a complex with other proteins such as RAD50 and NBS1 to participate in multiple nuclear DNA repair pathways (i.e.homologous end joining, nonhomologous end joining, and alternate non-homologous end joining).Hence, MRE11 is considered an nDNA maintenance protein.Previous studies has observed MRE11 localization to mitochondria and its binding to mtDNA.Those findings have motivated us to investigate the role of MRE11 in mitochondria where it may act as an mtDNA degrader of stalled replication forks.In this master's thesis, the effect of overexpression of MRE11 on mtDNA replication stalled by ddC (2′,3′-dideoxycytidine) in Flp-In T-REx 293 cells was analyzed using HincII nuclease digestion on 2D-AGE.In our study, we also determined the interactions of MRE11 exclusively in mitochondria.The protein of interest (MRE11) fused to a biotin ligase was targeted to mitochondria to biotinylate all proteins in proximity.Western blot was then probed against different replication and maintenance proteins such as Pol γ, TFAM, mtSSB, MGME1, RAD51c and TWNK, which are crucial for maintaining mtDNA.Our results identified that overexpression of MRE11 in Flp-In T-REx 293 cells did not affect replication fork stalling by ddCo.We also found no interaction between MRE11 and any of the proteins mentioned above.Therefore, our study suggest that MRE11 might play a limited role DNA replication and maintenance.
Author: Gondal, Waleed
Institution: University of Eastern Finland
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7024447819