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In eukaryotes, mitochondria are energy-producing organelles that are unique in containing their own genome, known as mitochondrial DNA (mtDNA). This multicopy DNA encodes 13 essential proteins that are fundamental to energy production. Consequently, defects in mtDNA can disrupt cellular energy processes, potentially leading to disorders ranging from mitochondrial diseases to cancers. Understanding the factors that protect mtDNA and maintain its stability is therefore crucial for ensuring efficient energy production and cellular health.

The incorporation of ribonucleotides (rNMPs) into DNA leads to severe genomic instability, including the formation of DNA breaks. Moreover, rNMP removal through topoisomerase 1 activity can produce harmful intermediates and generate short deletions within repetitive sequences. In this study, we investigate the endoribonuclease activity of mitochondrial topoisomerase 1 (hTop1mt) and compare it to that of its nuclear homolog, hTop1. Using various biochemical assays, we demonstrate that the mitochondrial enzyme exhibits a lower endoribonuclease activity than its nuclear counterpart, a difference that is partially explained by its weaker DNA-binding affinity and slower cleavage kinetics on linear DNA substrates containing an rNMP. Moreover, hTop1mt lacks the preference for cleaving at rNMPs that hTop1 exhibits. Consequently, on repetitive sequences, I showed that hTop1mt generates fewer rNMP-dependent deletions. These findings suggest that the potentially deleterious side effects of this rNMP repair pathway are minimized in mitochondria, providing further insight into why rNMPs in the mitochondrial genome are better tolerated than in the nuclear genome.

Mitochondrial single-stranded DNA-binding protein (mtSSB) is critical for maintaining mtDNA integrity during replication. Mutations in the SSBP1 gene, which encodes mtSSB, are frequently observed—being linked not only to mitochondrial diseases but also to cancer. However, the functional consequences of these cancer-associated mutations are not yet well understood. In this study, we investigated eight cancer-associated SSBP1 mutations through biochemical characterization. I showed that all mutant mtSSB proteins still form tetramers, but their DNA-binding capabilities and thermal stability was compromised. Collectively, these findings suggest that cancer-associated SSBP1 mutations can significantly impair mtSSB function, potentially leading to destabilization of mtDNA integrity.

Horizontal gene transfer between bacteria is closely linked to the spread of antibiotic resistance, and the type IV secretion system (T4SS) plays a crucial role in mediating this process. The pCF10 plasmid from the commensal bacterium Enterococcus faecalis encodes the machinery required for such transfer. In this study, we focused on characterizing the PrgE protein encoded by pCF10, which is proposed to function as a single-stranded DNA-binding (SSB) factor based on its sequence homology with known SSBs. In our structural analysis, we found that PrgE possesses the characteristic OB-fold typical of SSB proteins, yet it displays unusual DNA-binding properties. Specifically, we found that PrgE binds ssDNA very weakly and, surprisingly, exhibits a similar low affinity for dsDNA. In summary, PrgE is an OB-fold protein with atypical DNA interaction properties, and its precise role in the context of horizontal gene transfer remains to be fully elucidated.

In this thesis, I biochemically characterized three distinct DNA-binding proteins from different biological contexts. These findings not only deepen our understanding of the molecular functions of these proteins but could, hopefully, help in the development of novel therapies to enhance human health.