Director: Dr. Marie-Françoise Noirot-Gros

Research projects

In the cell, proteins execute their functions by interacting with other proteins, thus forming stable of transitory complexes. The identification of these interactions between proteins allows us to establish interaction maps, which underline the molecular machines and the regulation pathways connecting them. Our objectives are:

(i) To clarify the biological role of protein interactions involved in the control of replication initiation and elongation for the B. subtilis chromosome

(ii) To better understand the role of post-translational modifications in the regulation of these processes during the cellular cycle. To achieve these aims, we have developed an approach based on functional dissection involving the selective rupture of an interaction by mutation, the identification of the key amino acids for said interaction and the functional studying of these mutants in vivo and/or in vitro. This strategy allows mapping of the various interaction surfaces of a protein, as well as a better understanding of the mechanisms controlling the assembling of molecular machines in the cell. To top it all, our approach is a generic one and may therefore apply to any interacting protein couple, independently of the organism it derives from.

Main Undergoing Projects

Control of initiation and of its connections to the other cellular processes: a study of complexes associated with the DnaA initiator.

Chromosome replication in bacteria is a highly regulated process, in such a way that each origin of replication is initiated only once per cellular cycle. Within a interaction network centered around the initiator DnaA, we have identified the YabA protein as the first potential regulator of the initiation of replication in B. subtilis. Using a functional dissection strategy, we have established that YabA carried out its activity within a heterocomplex with DnaA and DnaN associated with the replication machinery during the major part of the cellular cycle. Several leads focused on the structural and functional characterisation of the YabA regulator, as well as the complex it forms with DnaA, are being followed to elucidate the control mechanism of intiation (BaSysBio). The functional dissection approach is also applied to the characterisation of the complexes formed by DnaA and two other partners: the membrane protein YkjA, whose function is unknown, and YqaH, a peptide coded by the integrated element “Skin”. We are investigating the role of these interactions in the control of DnaA’s activity during the cellular cycle (TranSys, BaSysBio).

Regulation of cellular processes by phosphorylation of proteins: Construction of an interaction network centered around a new family of bacterial tyrosin-kinases.

Bacterial kinases intervene in various aspects of the regulation of cellular processes, such as exo-polysaccaride synthesis, phage lysogeny or DNA metabolism. They are involved in the triggering of virulence pathways in pathogen bacteria (S. pneumoniae, S. aureus). Bacterial Tyrosin-kinases form a class of newly-identified enzymes whose role and mode of action within the cell remain uncharacterised. Their activity is triggered by an autophosphorylation, dependant itself on an interaction with a transmembrane modulator. In the framework of the ANR project “TyrPhosNet”, we are developing a protein-protein interaction network centered around tyrosin-kinases and their modulators in B. subtilis, using a two-hybrid approach. The exploration of this network through the functional dissection of tyrosin/modulators and tyrosin/substrates complexes should allow us to further our understanding of regulation patways and their coordination within the cell.

Functional duality of Flap-endonuclease activities in B. subtilis:

The DNA polymerase I in bacteria intervenes in the maturation of the Okazaki fragments during the replication of the “lagging” strand of the circular chromosome. This enzyme combines several activities, ensured by just as many functional domains, which consist of a DNA-dependant DNA polymerase, a 3’-5’ exonuclease activity responsible for the base edition activity and finally a 5’-3’ exonuclease function (Flap-endonuclease) involved in the processing of RNA primers. B. subtilis possesses a second 5’-3’ enxonuclease, YpcP, co-essential with the Pol I Flap-endonuclease activity. We interested in understanding the respective involvements of Pol I and YpcP in the replication of the “lagging” strand as well as in the stability of the B. subtilis chromosome.


Financings

European Projects:

• 

ANR:

• TyrPhosNet

Main External Collaborations

Partners for the TyrPhosNet Project :

• Pr. Ivan Mijakovic , bacterial Kinases (B. subtilis) (AgroPariTech, Micalis UMR1319)
• Dr . Christophe Grangeasse , tyrosine Kinase S. pneumoniae (IBCP, UMR 5086 CNRS/Université Lyon)
• Pr. Sysvie Nesler, structure-function of kinases/modulator complexes (LEBS, UPR 3082 CNRS, Gif)

Partners for the BaSysBio Project :

• Anthony Wilkinson, 3D structure of protein complexes (YSBL, University of York, UK)
• Frank Molina, model-structure of proteins (UMR3145 SysDiag/CNRS, Montpellier)
• MIG (UR1077);

Partners for the TranSys Project :

• Pr. Colin Robinson, System biology of membrane proteins (Warwick University, UK)
• Dr. JM van Dijl (University of Groningen, Netherland)

Individual Collaborations :

• Laurent Terradot , control of initiation in bacteria (IBCP, UMR 5086 CNRS/Université Lyon)
• J-P Claverys (LMGM, UMR5100, CNRS/Université Toulouse II)
• Sophie Quevillon-Cheruel (IBBMC, UMR 8619 CNRS-Université Paris-Sud 11)
• Alain Trubuil, analysis of bacterial protein intracellular dynamics ( MIA, UR341, INRA Jouy)