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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.

Mirin, a chemical inhibitor of MRE11, has been recently reported to suppress immune response triggered by mitochondrial DNA (mtDNA) breakage and release during replication stalling. We show that while Mirin reduces mitochondrial replication fork breakage in mitochondrial 3´-exonuclease MGME1 deficient cells, this effect occurs independently of MRE11. We also discovered that Mirin directly inhibits cellular immune responses, as shown by its suppression of STAT1 phosphorylation in Poly (I:C)-treated cells. Furthermore, Mirin also altered mtDNA supercoiling and accumulation of hemicatenated replication termination intermediates-hallmarks of topoisomerase dysfunction-while mitigating topological changes induced by the overexpression of mitochondrial TOP3A, including TOP3A-dependent strand breakage at the noncoding region of mtDNA. Although Mirin does not seem to inhibit TOP3A activity in vitro, our findings demonstrate its MRE11-independent effects in cells and give insight into the mechanisms of the maintenance of mtDNA integrity.

The incorporation of ribonucleotides (rNMPs) into the nuclear genome leads to severe genomic instability, including strand breaks and short 2-5 bp deletions at repetitive sequences. Curiously, the detrimental effects of rNMPs are not observed for the human mitochondrial genome (mtDNA) that typically contains several rNMPs per molecule. Given that the nuclear genome instability phenotype is dependent on the activity of the nuclear topoisomerase 1 enzyme (hTOP1), and mammalian mitochondria contain a dis]nct topoisomerase 1 paralog (hTOP1MT), we hypothesized that the differential effects of rNMPs on the two genomes may reflect divergent properties of the two cellular topoisomerase 1 enzymes. Here, we characterized the endoribonuclease activity of hTOP1MT and found it to be less efficient than that of its nuclear counterpart, a finding that was partly explained by its weaker affinity for its DNA substrate. Moreover, while hTOP1 and yeast TOP1 were able to cleave at an rNMP located even outside of the consensus cleavage site, hTOP1MT showed no such preference for rNMPs. As a consequence, hTOP1MT was inefficient at producing the short rNMP-dependent dele]ons that are characteristic of TOP1-driven genome instability. These findings help explain the tolerance of rNMPs in the mitochondrial genome

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

The mitochondrial single-stranded DNA-binding protein (SSBP1) is critical for mitochondrial DNA (mtDNA) replication and stability. Mutations in SSBP1 have been linked to mitochondrial disease and are found in cancer samples, but the functional consequences of the cancer- associated SSBP1 mutations remain poorly understood. This study investigates eight cancer- associated SSBP1 mutations by assessing their effects on SSBP1’s DNA binding, thermal stability, and tetramerization. We found that the tetramerization remained unaffected across all mutants. However, all mutant proteins exhibited reduced DNA binding affinity, with significant defects observed for R91W, P43S, and D77Y. Thermal stability was also compromised in most variants tested, particularly R38Q, S29F, and R91W, as evidenced by reduced melting temperatures. These findings suggest that cancer-associated mutations compromise protein stability and impair SSBP1’s ability to stabilize single-stranded DNA, potentially disrupting its role in mtDNA maintenance and contributing to mtDNA instability in cancer cells.