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Structural Studies Of Human Ner Multi Protein Complexes Of The Ner Pathway: How Do Cells Eliminate Dna Damages Caused By Platin-Based Drugs Used In Cancer Chemotherapy?

Reference : PhD Arnaud POTERSZMAN

Offer publication : April 30, 2016

Responses to DNA damage are essential for the maintenance of the genome integrity and alteration of repair mechanisms have a
causal role in the development of cancers. This project focuses on the nucleotide excision repair (NER) pathway which corrects UV induced lesions as well as DNA-damages that result from drugs used in chemotherapy such as cis-platin derivatives.

More precisely, this project focuses on the transcription/DNA repair complex TFIIH, a multi-protein complex composed of 10 subunits that harbours three enzymatic activities and can be
resolved into two functional and structural entities: the 6-subunits core-TFIIH with the XPB helicase and the 3-subunits Cdk Activated Kinase complex (CAK). The XPD helicase bridges core-TFIIH to CAK. Initially identified as basal transcription factor, TFIIH also participates in the transactivation of several hormone-dependent genes by phosphorylating nuclear receptors and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites and recruitment of additional repair factors.

Following recent work in the team, production of recombinant core-TFIIH as well as CAK and XPD in quantity and quality sufficient for structural studies has been setup. The work proposed here has two main related. The first one is to understand is to determine the structure of core-TFIIH and of
higher order complexes such as Holo-TFIIH or TFIIH/XPG by cryo-electron microscopy (cryo-EM). These data will be combined with biophysical studies and mass spectrometry analysis (chemical cross-linking) to unravel the protein-protein interaction network within core-TFIIH and provide new
insights into the XPB subunit which is the target of triptolide (TP), a natural molecule with anti-inflammatory and anticancer activities.

To better understand how TP inhibits XPB and therefore transcription and NER, the project will also include crystallization of the catalytic XPB ATPase domain in the presence of TP, as a basis for structure/function studies. The second aspect of the work will focus on TFIIH in the context of the NER reaction to investigate how TFIIH is loaded onto the damaged DNA and
better understand its role in the assembly of the pre-initiation complex. DNA fragment containing a single cis-platin damage located in the middle of the sequence will be used to prepare stable complexes that involve TFIIH for cross-link-MS analysis and structure determination by cryo-EM. Combined with in vitro functional studies these data will help to better understand how TFIIH is recruited by the damage recognition complex XPC/HR23B, how, via its XPB and XPD activities TFIIH promotes the assembly of the pre-initiation complex and converts a damaged site into a substrate for the XPF and XPG endonucleases

A number of DNA damaging used in chemotherapy, among which cis-platinum, lead to the formation of bulky adducts that are repaired by NER. For certain cancers, it is accepted that the capacity of cells to be repaired is inversely correlated to chances of success, and one can expect that inhibition of NER should increase the efficiency of the treatment. To develop new methods and drugs capable of interfering with NER, structure-function studies of its components are thus essential.

- WISHED SKILLS : Basal education in molecular, cellular and/or structural biology as well as a strong motivation for fundamental questions in biology are required.

- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : Production and purification of multi-protein complexes using prokaryotic and eukaryotic expression systems as well as stable cell lines. Characterization of complexes by biochemical (immuno-precipitations, gel-shift assays, SPR, …) and biophysical (gel-filtration, light scattering, analytical ultracentrifugation, SANS, SAX…) methods. Structure determination by cryo-EM, structure/function relationships.


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Application Deadline : Dec. 31, 2016

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