Prokaryotic DNA repair systems: mechanistic characterization and valuable insights for biotechnological applications

This thesis aimed at characterizing DNA repair systems in bacteria. We provided new insights for the description of the Nucleotide Excision Repair (NER) system, contributing to elucidate molecular details still unclear about the pathway. On the other hand, we applied biotechnological engineering to exploit Direct Damage Reversal in order to develop useful tools that can be used to improve research outcomes. Concerning the NER project, we were able to describe additional features of the damage recognition process in Mycobacterium tuberculosis, carried out by UvrA and UvrB proteins. We conducted Cryo-EM based structural studies, obtaining four models depicting different snapshots of the dynamic damage recognition process. We were able to suggest a new mechanism of the damage recognition process, with UvrA being essential for damage identification. These results led to a publication, currently accepted in Nature Communications. Then, in collaboration with Vrije Universiteit of Amsterdam, we exploited optical tweezers and single-molecule fluorescence imaging to collect some preliminary results, indicating a different mechanism of action of UvrA towards damaged and undamaged DNA, in terms of forces applied onto the DNA molecules, movement and interaction time. Besides, we developed a new self-labeling protein tag (SLP) suitable for studies on (hyper)thermophilic organisms, by applying an integrated computational and structural approach, obtaining an engineered variant of Saccharolobus solfataricus OGT (SsOGT) able to recognize O6-benzyl-cytosine derivatives, to be used in non-permissive reaction conditions where commercial SLPs fail. The project led to a paper published in 2022 in Computational and Structural Biotechnology Journal. Starting from this technology, we employed the OGT to develop molecular rulers to measure distances between organelles in living cells. The method revealed to be specific, precise and efficient, and led to the deposition of a patent.