We study the molecular mechanisms involved in Genome Replication, Integrity and Repair
using techniques mainly from Structural and Molecular Biology and also Computational Biology.
We can venture also into topics such as Synthetic and Systems Biology.
We are part of the Department of Structural Biology and Chemistry in Institut Pasteur
with a secondary affiliation with the Department of Computational Biology.
We use experimental techniques such as crystallography and cryo-electron microscopy to visualize at the atomic level
the structure of molecules essential to life and to understand their functional properties, especially for:
• DNA and RNA polymerases involved in genome replication and other transactions (repair, transcription, recombination...)
• Ion channels involved in electric nerve signaling and cell-cell communications.
We complement them with computational approaches such as molecular dynamics (atomic models),
normal modes dynamics (coarse-grained models), conditioned Langevin dynamics
in order to go beyond the essentially static pictures given by these methods.
We are interested into generating the structures of intermediates along a transition path between two known conformational states of very large macromolecular assemblies.
In addition, we use physics-based approaches such as statistical thermodynamics
to make use of the information contained in biodiversity.
The massive sequence data of related molecules in the tree of life can help to understand
what is essential in their active site structure and how it is modulated (allostery).
Our main goal is to understand how these molecular machines interact with partners at the atomic level, their solvation and binding properties (including electrostatics),
so as to design structure-inspired drugs (pharmacology and drug discovery).
When possible we study their structure in the context of their partners in larger
macromolecular complexes in order to mimic their behaviour in cellulo
and to understand their regulation as well as their emerging collective properties (systems biology).
Protein engineering: Another aim is to make DNA polymerases evolve by directed evolution techniques
to accept other substrates such as NTPs (RNA polymerases) or XTPs (xenonucleotides) or different templates (reverse transcriptases).
One source of inspiration is phage genomes containing hypermodified bases (synthetic biology).
AI methods can be used to further understand large data sets of the sequences of the evolved molecules.
Left:
DNA polymerases are essential enzymes involved in DNA Duplication, Rearrangements, and Repair - when it is damaged.
They are present in all branches of life and have been classified into different Families based on their sequences.
Table 1 (Patel and Loeb, NSMB, 2001) shows all known classes of DNA Polymerases; the last one is now called Family Y.
Family A and B share in fact 3 common sequence motifs and
have the same catalytic site and general folding-type called the Klenow-fold, also shared by RT (Delarue et al., Prot. Engng, 1990).
Family Y also shares the Klenow fold.
Family C is the main replicative DNA polymerase (bacterial pol III) and has the same fold as PolX, involved in DNA Repair (nucleotidyltransferase fold).
Finally PolD has a different fold, common with RNA Polymerases, known as the double-psi-beta-barrel fold (Sauguet et al., Nature Commun. 2016).
The structure shown is Terminal deoxynucleotidyltransferase (TdT), a polX, caught in the act of repairing a double-strand break in the DNA.
Right: Xtal structure of a pentameric ligand-gated ion channel called GLIC in the presence of a general anesthetics (Nury et al., Nature 2011) in a lipidic environment. On top, a typical crystal is shown, as well as its diffraction pattern.
We study the molecular mechanism of DNA Repair for Double Strand Breaks by the Non-Homologous End Joining (NHEJ) process in eukaryotes using x-ray crystallography and cryo-EM, especially steps involving pol mu or TdT
We work with archaeal (PolB) and bacterial thermophilic (polA) DNA polymerases to change their specificity by directed evolution techniques, using structural information to guide the design of libraries.
We study the general architecture of the replisome in archaea, especially around the essential PolD, that we discovered to be unique among other DNA polymerases, as it has the fold of multi-subunit RNA polymerases.
We are developing new computational methods to calculate the electrostatics of proteins, understand their dynamical properties and simulate transitions between two known conformations of the same macromolecule.
We study the structure and function of ligand-gated ion channels by X-ray crystallography to understand
• the gating mechanism (opening of the pore upon agonist binding)
• the permeation mechanism (transport of ions through the pore)
• modulation by allosteric compounds (general anesthetics, barbiturates, amphetamines …)