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The increase of antibiotic resistance is a major threat to human health. The spread of mobile genetic elements (MGEs) via conjugation is a major contributor to this problem, especially in hospital settings. Many MGEs encode Type IV Secretion Systems (T4SSs), which are multiprotein complexes that transfer the MGE from donor to recipient cells. T4SSs are versatile systems that exist in all prokaryotes. While most research has focused on T4SSs from Gram negative (G^–) bacteria, it is important to understand the similarities and differences with T4SSs from Gram positive (G⁺) bacteria, given their different cell envelopes. Additionally, there is also variability within G^– T4SSs, which is not yet fully understood.

The aim of this thesis was to explore the diversity of T4SSs, using pKM101 from E. coli (G^–) and pCF10 from E. faecalis (G⁺) as model systems, with a focus on DNA transfer and replication (Dtr) proteins.

We biochemically characterized the relaxase TraI from pKM101, which processes plasmid DNA prior to transfer through the T4SS. We also solved the crystal structure of its transesterase domain with and without its substrate oriT DNA, highlighting its conserved mechanism of action. We further explored the relationship between TraI and the accessory protein TraK, using AlphaFold to predict an interaction involving the TraI CTD. This was confirmed experimentally using in vivo BPA-crosslinking.

Many conjugative plasmids encode single-stranded DNA-binding proteins (SSBs), which are thought to protect DNA during transfer. pCF10 encodes the protein PrgE, which was proposed to be one such SSB. However, our biochemical studies and X-ray crystallography revealed that PrgE is an OB-fold protein with unexpected DNA-binding behavior. While its benefit for the plasmid remains unclear, our functional studies have shown that it does not play a role in conjugation.

Finally, we analyzed the structural diversity of conjugative T4SSs in G^– and G⁺ bacteria, using bioinformatics and structural modelling. This revealed unknown commonalities, which indicate that G⁺ T4SS mating channels are likely more similar in structure to G^– T4SSs than expected.

In summary, this thesis provides new insights into the Dtr proteins that play an integral role in T4SS mediated conjugation, knowledge that hopefully can be used in the fight against hospital acquired infections in the future.

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.