BESE invited seminar - Prof. Gabriel Moncalian Montes

5/10/2017 10:00 AM - 5/10/2017 11:00 AM
Relaxases (also named Mob proteins) are enzymes involved in DNA processing for plasmid conjugation. Relaxases are usually multidomain proteins, being the relaxase domain always located at the N-terminal position. According to the phylogeny of the relaxase domain, conjugative transfer systems have been classified in six MOB families: MOBF, MOBH, MOBQ, MOBC, MOBP and MOBV. The MOBF, MOBQ, MOBP and MOBV relaxases belong to the HUH endonuclease family while the other two families are unrelated and insufficiently characterized. HUH proteins are enzymes widespread in all three domains of life, where they process DNA during initiation of rolling circle replication (RCR) of certain phages and eukaryotic viruses, conjugative transfer of plasmid between cells and transposition of insertion sequences and helitrons. These enzymes contain an HUH motif, in which two conserved histidines (H) involved in metal coordination are separated by a hydrophobic residue (U). HUH endonucleases also contain a Y motif, which contains the tyrosine(s) involved in the nucleophilic attack to cleave and rejoin the target single strand DNA (ssDNA). One attractive feature of HUH proteins is that the nucleophilic attack on specific ssDNA sequences results in stable protein-DNA covalent linkages. Thus, HUH proteins provide a tool for site specific bioconjugation of proteins to ssDNA, for instance to DNA origami nanostructures, where ssDNA is folded into a desired shape with the aid of hundreds of oligonucleotides named “staples”. Relaxases such as TrwC of plasmid R388 or TraI of plasmid F have been proven to form protein-DNA conjugates efficiently. Using this approach, DNA can be conjugated to any desired protein fused to either the N-terminus or the C-terminus of recombinant relaxases, without losing activity. Moreover, HUH proteins exhibit site-specific recombination and have been used as an efficient strategy for genome editing. The replicase of Adeno-associate virus (AAV) and relaxase TrwC from plasmid R388 catalyze site-specific DNA integration into human genomes and thus constitute potential new tools for genome editing.​