Description du sujet de thèse

Mismatches (MM) between two DNA bases can i) occur during DNA replication, ii) are formed by heteroduplex formation during homologous recombination (HR), or iii) result from the action of mutagenic chemicals, ionizing radiation or spontaneous deamination (1). Although efficient mismatch repair (MMR) prevents transmission of mutations between generations, unrepaired MMs also underpin genetic diversity, adaptation, and speciation of microbial populations. In the case of MM formation during recombination (homologous recombination), the MMR prevents homologous recombination between non-identical sequences and thus contributes to the genetic interspecies barrier and evolution of species (2).
Earlier bioinformatics studies failed to identify the canonical MMR repair genes mutSL in many archaeal and bacterial genomes. However, a combination of genetic and biochemical studies led to the discovery of a non-canonical mismatch-specific endonuclease NucS (also dubbed EndoMS), which is frequently found in actinobacteria (including mycobacteria) and archaea. The objective of the present multidisciplinary project is now to study the mechanisms and function of this novel mismatch repair system (MMR).
It has been shown that this novel path of repair uses the endonuclease activity of the proteins NucS/EndoMS to create a break in the ADNdb around the mismatch (3). Here we aim at analyzing in detail the structural arrangement of the protein-ADN complexes involving the molecular actors of this original repair process. The recombinant proteins will be produced in Escherichia coli and purified by chromatographic methods. Subsequently, several complementary structural methods, such as small angle X-ray scattering, crystallography and also electron cryo-microscopy, will be applied to study the formed complexes in solution and in the crystal. The production and purification of the protein actors (NucS et PCNA) of the repair system are already mastered for the Pyrococcus abyssi proteins, and we will screen for different DNA "mismatch" (or MM) substrates, branched or not, in the aim of identifying the DNA length and stoichiometry of actors that lead to the repair complex formation. these structural studies are part of the larger ANR collaborative project (MMRDNABREAK), that combines biochemical, genetic and structural biology approaches to understand in more detail how the genetic information to repair MMs is restored, from intact matrix by homologous recombination, or by non-homologous end-junction repair, which frequently introduces errors. The project "MMRDNABREAK" will help us understand the main differences between the two repair paths that allow detecting and repairing mismatches, which are frequently introduced during replication or recombination of DNA. Since these repair paths are only present in archaea and actinobacteria, these proteins potentially also represent new targets to be inhibited for the development of alternative antibiotics, and/or represent novel biotechnological tools to engineer DNA.
The activity of the candidate will be to produce and purify lthe proteins and ternary complexes (NucS-PCNA-MM-DNA), and to manage the biophysical analyses necessary to characterize and structurally analyse these complexes. The activites can include protein crystallography and analyses using SAXS and/or cryo-EM.
1. Kunkel, T.A. and Erie, D.A. (2015) Annu Rev Genet, 49, 291-313
2. Spies, M. and Fishel, R. (2015) Cold Spring Harb Perspect Biol, 7, a022657.
3. Creze, C., Ligabue, A., Laurent, S., Lestini, R., Laptenok, S.P., Khun, J., Vos, M.H., Czjzek, M., Myllykallio, H. and Flament, D. (2012). J Biol Chem, 287, 15648-15660.

Contexte de travail

The doctoral contract is co-financed by the ANR project MMRDNABREAK, coordinated by Dr. Hannu Myllykallio at école Polytechnique Paris, and the Région Bretagne (program ARED 2023), in the collaborative context between three laboratories: the 'Laboratoire d'optiques et biosciences', CNRS UMR 7645-INSERM-Ecole Polytechnique, the laboratory LM2E, UMR 6197, IFREMER-UBO-CNRS and the 'Station Biologique de Roscoff', LBI2M, CNRS-UMR8227-Sorbonne Université. The contract work will be situated in the CNRS laboratory LBI2M, partner of the ANR project, and specifically in the group of Marine Glycobiology of the UMR8227, and at the crystallographic platform of the Station Biologique de Roscoff. Internships, at the LM2E for protein biochemistry (production, purification), and at the école Polytechnique for the electron cryo-microscopy studies will also be programed. The host institute, the 'Station Biologique de Roscoff', is a public research center in marine biology and oceanology located in the city of Roscoff on the north cost of Brittany. Within the « Laboratoire de Biologie Intégrative des Modèles Marins » (LBI2M), the research of the Marine Glycobiology group is focused on the structure-function relationship of marine enzymes active on polysaccharides in general. The project includes the resolution of structures by crystallography at the SOLEIL synchrotron in St Auban, and short-term visits traveling to the synchrotron will be possible.

Contraintes et risques

Constraints are linked to manipulating in a laboratory with biochemical, as well as molecular and structural biology tools.

Informations complémentaires

The candidate should have a valid master degree in biochemistry and structural biology of proteins.
The candidate should have solid knowledge about at least one method in structural biology, and in heterologous production and purification of proteins. Knowledge about the molecular mechanisms of DNA-repair systems would be of advantage. In addition, the candidate should have the skills to formulate and lead a scientific project, to communicate and valorize the work, should fluently master english at all levels (read, write and speak), and should have all skills to interact and work within a group.