Structural And Functional Studies Of Human Topoisomerases Complexes With Cancer Chemotherapy Compounds
Reference : PhD Valérie LAMOUR
Offer publication : April 6, 2016
Type 2 DNA topoisomerases (Top2) control DNA topology during replication, transcription and chromosome segregation. Essential for cell proliferation, these enzymes are targets for several classes of antibacterial and antineoplastic compounds of prime importance for human health. The emergence of antibiotic resistant bacterial strains and the selection of resistance clones during cancer treatment urge for the development of new
drugs and for the better understanding of their mode of action. Structural research in the Top2 field has, to date, been limited to individual domains that do not take into account the overall conformation of the enzyme adopted during their catalytic cycle. Moreover, there is only scarce information on the cellular complexes associated to Top2 enzymes and targeted by drugs.
This thesis proposal aims at the functional and structural study of human DNA topoisomerases to provide knowledge on the enzymatic mechanisms and on drug targeted Top2- associated cellular complexes. The human Top2a and Top2b isoforms share high sequence conservation and a similar structural organization with the bacterial enzyme. Top2 is a biomarker
for cancer and a key chemotherapy target for anti-tumour drugs such as etoposide. The Top2 functional dimer has a size of over 340kDa. Previous studies on the bacterial DNA topoisomerase allowed us to overcome the technical challenges and establish the adequate protocols for dealing with such large, modular macromolecular assemblies (Papillon et al, NAR 2013). A production protocol for the human enzymes has been successfully setup in yeast and enzymatic and functional tests are routinely implemented in the laboratory. The structural study by electron microscopy is ongoing on a full length human Top2, in complex with DNA and anti-cancer molecules. We obtained preliminary electron microscopy data that will be immediately useful from the start of the thesis project. We have designed alternative DNA templates sensitive to anti-cancer drugs that will be used to stabilize further the existing complex.
This first reconstruction of a full length human Top2 will be used as a platform for studying larger assemblies with cellular partners. Identified interactions with transcription factors centered on Top2 in the context of chromatin transcription complexes will be investigated such as nuclear receptors for which we can benefit from purified recombinant proteins in the department. Structural techniques (cryo-electron microscopy, x-ray crystallography, etc) and biophysical approaches are used in combination with functional studies to gain insight into the tridimensional architecture of Top2-associated complexes. This thesis will take place in the Integrative Structural Biology
Department (IGBMC) in the team of V. Lamour/M Ruff. The student will benefit from the technical support of engineers in biochemistry and electron microscopy. A collaboration for the functional aspects of this study has been set up with the team of Carolin Austin (Univ Newcastle, UK) that is working on cancer therapy targeting the Top2 enzymes. In the frame of our projects we also collaborate with organic chemistry and mass spectrometry laboratories in Strasbourg.
- WISHED SKILLS : The PhD candidate must have a Master degree in Biology, ideally in Structural Biology, biophysics or chemical biology with basic knowledge in biochemistry and structural techniques such as x-ray crystallography and electron microscopy. He/she must have a strong interest and motivation for research at the interface between biology, chemistry and physics. Knowledge of scientific english and a capacity to work within a team environment are a prerequisite.
- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : The PhD student will be trained in biochemistry (medium scale protein purification, pulldown experiments, basic enzymology and molecular biology tools), in structural biology techniques (electron microscopy and xray crystallography) and other biophysical techniques depending on the project development. The student will get the opportunity to present his/her work through departement and institute seminars, will be encouraged to attend international conferences and workshops including in the context of the “Ecole Doctorale des Sciences de la Vie” and the IGBMC PhD program.
Application Deadline : Dec. 31, 2016