Biochemical and structural characterization of proteins involved in prokaryotic DNA metabolism as source of novel drug targets and biotechnological solutions

The aim of the presented PhD project was the biochemical and structural characterization of proteins and enzymes involved in prokaryotic DNA metabolism as potential target for antitubercular drug discovery as well as for the design of biotechnological tools for application in molecular biology. A variant of the 06-DNA-alkyl-guanine-DNA-alkyl-transferases (OGT) from Saccharolobus solfataricus (HS) has been proposed as a thermostable self-labelling protein tag in order to expand this technology to harsh experimental conditions and extreme environments. We present here the crystal structure of H5 protein in complex with the fluorescent probe functional to a structure based protein engineering aiming at further evolution of the proposed technology based on a chemo-enzymatic approach for the labelling reaction. The second part of the presented thesis focused on the characterization of protein complexes from Mycobacterium tuberculosis (MTB) involved in the Nucleotide Excision Repair (NER) pathway and the initiation phase of DNA replication. To this aim we obtained a low-resolution model of the UvrA-UvrB complex acting in the damage recognition step along NER pathway. The structural model allow us to map three residues involved in protein-protein interaction whose key role was further confirmed by site directed mutagenesis experiments. On the other hands, we obtained preliminary results on the biochemical characterization of a novel helicase loader acting during DNA replication in MTB, namely DciA. In order to shed light on mechanistic aspects of DciA-dependent helicase in MTB, we investigated the secondary structure element of DciA domains protein using limited proteolysis coupled with native mass spectrometry. Moreover, we determined the kinetic constants of the interaction between DciA loader and the target helicase by means of Surface Plasmon Resonance.