TOPCANBIOX · Structural and functional studies of eukaryotic and prokaryotic topoisomerases and their complexes with molecules of therapeutical interest

Structural and functional studies of eukaryotic and prokaryotic topoisomerases and their complexes with molecules of therapeutical interest

Final Publishable summary report Objective N°1- Structural studies of a full length prokaryotic topoisomerase II in complex with a molecule of therapeutic interest We have set up the overproduction and large-scale purification tools for the 320 kDa bacterial topoisomerase II from the start of this project. In vitro enzymatic and functional assays were optimised to characterize the recombinant enzyme. The success of our project was bound to our ability to stabilize a defined conformation of the enzyme. Macromolecular complexes such as DNA/protein and DNA/protein-small molecule have been characterised using a combination of biochemical, biophysical and mass spectrometry analysis through a collaborative work with a mass spectrometry laboratory in Strasbourg, finally leading to structural studies by cryoelectron microscopy in the IGBMC Structural Biology Department. We were able to determine, for the first time in the field, the full architecture of this type II DNA topoisomerase, alone and bound to a 155bp DNA sequence and an antibiotic. These results show (i) an unexpected structural arrangement of the enzyme subunits that has consequences on DNA crossover selection during DNA relaxation for all type II DNA topoisomerases and (ii) the structural basis of DNA wrapping during DNA supercoiling by DNA gyrase. A publication has been submitted for publication mid-June 2012. We also have initiated a complementary approach to topoisomerase-associated complex identification through chemical proteomics in collaboration with chemists at the Strasbourg Faculty of Pharmacy. Two articles have been published (Budin et al., ChemBiochem 2010) and the identification of DNA topoisomerase antibiotic associated complexes is under way. This study sets up the grounds for the perspective project of TOPCANBIOX aiming at structural studies of larger macromolecular complexes associated with topoisomerases. Tasks achieved for the reporting period - Overproduction and large-scale purification tools for the 320 kDa bacterial topoisomerase II (DNA gyrase) and implementation of in vitro enzymatic and functional assays - Identification of potential new inhibitors in collaboration with chemists - biochemical characterisation under way - Determination of a full length DNA- protein and DNA-protein-small molecule cryoEM structure with complementary biochemical and supramolecular mass spectrometry data - article submitted - Initiation of a transversal approach in collaboration with chemists aiming at the identification of topoisomerase-associated complexes using chemical proteomics - article in preparation on the novobiocin antibiotic secondary targets - initial structural study of one identified supramolecular complex centered on the bacterial topoisomerase Initial data and procedures established on the bacterial enzymes are now transposed to the study of the human topoisomerases, targets for anti-cancer drugs. This work funded by the Marie Curie (MC) grant has produced outstanding results that will be soon published and has allowed to establish a long lasting research project on the structural studies of DNA topoisomerases complexes. Objective N°2- Functional and Structural studies of eukaryotic topoisomerase II? and its interaction with chemotherapeutic agents. We have established the overproduction in yeast and purification of human topoisomerase II a and ß isoforms by modifying yeast expression vectors encoding for the two proteins. We have optimised the in vitro enzymatic and functional assays of the prokaryotic enzyme for the homologous human enzymes. Complexes of the full length enzymes are being studied with various biochemical and biophysical techniques prior to structural analysis. We have initiated the structural studies by cryoelectron microscopy of the full length enzymes based on our successful procedures with the bacterial enzymes. We are also developping chemical probes based on anti-cancer therapeutic molecules in collaboration with chemists as we did for the bacterial enzymes. These probes will be used to perform pulldowns on cancer cell lines to identify human Topoisomerase II interacting complexes targeted by these drugs. Tasks achieved for the reporting period - Overproduction in yeast and purification of human topoisomerase II a and ß isoforms - Implementation of in vitro enzymatic and functional assays. - Initiation of mass spectrometry studies of protein-DNA complexes with small molecules. - Initiation of full length enzymes structural studies by cryo-electron microscopy - Chemical proteomic experiments using probes derived from anti-cancer drugs targeting human topoisomerases, for which additional funding has just been obtained early 2012. Reintegration objective The reintegration objectives as stated in the MC international reintegration grant (IRG) were already fully met for the mid-term reporting period. Thanks to the MC IRG support, Valerie Lamour was able to establish her research at the internationally renowned biomedical research institute IGBMC (France) in the structural biology departement. Three young researchers have joined Valerie Lamour research project: a PhD student supported by a French CNRS/Alsace region fellowship (2008), two engineers successively hired thanks to the MC reintegration funding and a second PhD student from Strasbourg University. Additional funding has been obtained early 2012 to maintain our established expertise through an engineer for another two years, in the continuity of the MC grant. The recognition of Valerie Lamour's research potential impact on human health through the MC FP7 european program contributed to her success in getting a tenured position and she was ranked first for a competitive CNRS CR1 position in 2009 and the same year was also offered a position with the faculty of medicine. She chose to join Strasbourg University in September 2009 as an assistant professor attached to the University Hospitals to position her scientific career at the interface between fundamental and clinical research and is now coordinator of one of the few MD-PhD program in France. Valerie Lamour has just been promoted to class I assistant professor in June 2012. Please see Annex 1

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