Understanding the Mechanism of Double strand Break Repair and Genome Fragility in Mammalian Mitochondria

Abstract

Mitochondria are semiautonomous organelles in a cell as they have their own genome that replicates independently. It plays a major role in oxidative phosphorylation due to which mitochondrial DNA (mtDNA) is frequently exposed to oxidative damages. Factors such as ionizing radiation, radiomimetic drugs and replication fork stalling can lead to point mutations, deletions and other rearrangements resulting in mitochondrial genome fragility. Since a distinct feature of the mitochondrial genome is the absence of noncoding regions, mutations can severely affect the mitochondrial functions. In many cases, deletions in human mtDNA are flanked by short direct repeats; however, the mechanism by which they arise is still unknown. Mitochondria from patient samples show frequent genomic aberrations such as point mutations, insertions and large-scale deletions that could possibly account for mitochondria associated disease pathogenesis including cancer. Previous studies have shown the association of several mitochondrial mutations and deletions with ageing and human disorders such as myopathies, dystonia and hepatocellular carcinoma. Bioinformatic analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority (87%) are located at G4 DNA motifs. Interestingly, among this, ~50% of the break points were due to a deletion at base pair position 8271-8281, ~35% were due to deletion at 12362-12384 and ~12% due to deletion at 15516-15545. In first part of my thesis, I have investigated the molecular basis for the occurrence of mitochondrial DNA deletions. Firstly, different non-B DNA structure forming motifs were investigated in mitochondrial genome by using non-B DNA prediction tools such as non-B DB database and QGRS mapper. Results revealed the presence of five G-quadruplex forming motifs and several inverted and direct repeats. Formation of G-quadruplex DNA structures at the mitochondrial fragile regions were characterized by using various biochemical assays like electromobility shift as