Laboratoire d'Optique et Biosciences

Latifa BOUZHIR-SIMA, INSERM

Joëlle KUHN, CNRS

Isabelle LAMARRE-JOUENNE, INSERM

Tamara BASTA-LE BERRE (ANR)

Roxanne LESTINI (ANR)

Our research aims to improve our understanding of the mechanisms of molecular regulation and adaptation, notably in prokaryotic systems.

1) Bacterial sensor proteins, essential components of bacterial signal transduction chains in adaptation to hypoxic conditions. We are trying to obtain structure-based dynamic information concerning the binding to and the release of oxygen from the protein sensor domain, as well as to get insight into the mechanisms which correspond to "functioning" of these sensors. A part of these studies is done in close synergy with M. Vos (see «Internal protein dynamics»), using an approach associating molecular biological and biochemical methods with ultrafast absorption and Raman spectroscopy. We also try to understand the mechanism of coupling between oxygen binding to the sensor domain and signal transduction in the holoprotein (UL).

2) The reaction mechanism and metabolic regulation of a new class of thymidylate synthases, ThyX. Our particular interest focuses on dissecting the rate limiting step(s) of ThyX catalysis and on investigating the novel links of folate and thymidylate metabolism in ThyX-containing species, especially in the human pathogen Helicobacter pylori. Moreover, ThyX enzymes, present in many pathogenic Bacteria, but absent in humans, represent an attractive target for specifically inhibiting microbial growth. Another project concerns optimizing very recently identified inhibitors of ThyX towards possible biomedical applications (HM, UL).

3) Moreover, we are interested in the identification and investigation of novel protein complexes required for genome maintenance in micro-organisms (particularly archaea), thriving at conditions approaching the physical limits of life. In particular, we are attempting to understand how the interplay of DNA replication and repair processes ensures the maintenance of genomic integrity. This research is motivated by the belief that the discovery and investigation of gene and protein function at molecular level using new experimental approaches is a key aspect of the post-genomic era (HM, HB).

Technical approaches
In silico methods and phylogenomics, microbiology and cellular biology, molecular biology, bioengineering, protein biochemistry and enzymology, inhibitory screening, associated spectroscopic techniques including time-resolved FRET analyses.

Technology offers

·  ThyX, a novel thymidylate synthase and related inhibitors (Oseo and INSERM Transfer - Bourse de technologies)
·  Putting together the Okazaki fragments (European Union, Cordis Technology offer and marketing agreement)

Selected publications  
1.  Lechauve C, Bouzhir-Sima L, Yamashita T, Marden MC, Vos MH, Liebl U, Kiger L (2009)  
Heme ligand binding properties and intradimer interactions in the full-length sensor protein dos from Escherichia coli and its isolated heme domain.
J Biol Chem 284(52): 36146-36159 
 
2.  Ren B, Kuhn J, Meslet-Cladiere L, Briffotaux J, Norais C, Lavigne R, Flament D, Ladenstein R, Myllykallio H (2009)
Structure and function of a novel endonuclease acting on branched DNA substrates.  
EMBO J 28(16): 2479-2489 

3.  Escartin F, Skouloubris S, Liebl U, Myllykallio H (2008)
Flavin-dependent thymidylate synthase X limits chromosomal DNA replication.
Proc Natl Acad Sci U S A 105(29): 9948-9952

4.  Yamashita T, Bouzhir-Sima L, Lambry JC, Liebl U, Vos MH (2008)
Ligand dynamics and early signaling events in the heme domain of the sensor protein Dos from Escherichia coli.
J Biol Chem 283(4): 2344-2352
  
5.  Brooks MA, Meslet-Cladiere L, Graille M, Kuhn J, Blondeau K, Myllykallio H, van Tilbeurgh H (2008)
The structure of an archaeal homodimeric ligase which has RNA circularization activity.
Protein Sci 17(8): 1336-1345.  

6.  Meslet-Cladiere L, Norais C, Kuhn J, Briffotaux J, Sloostra JW, Ferrari E, Hubscher U, Flament D, Myllykallio H (2007)
A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen.  
J Mol Biol 372(5): 1137-1148  

7.  Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H, Allers T (2007)
Genetic and physical mapping of DNA replication origins in Haloferax volcanii.
PLoS Genet 3(5): e77  

8.  Graziani S, Bernauer J, Skouloubris S, Graille M, Zhou CZ, Marchand C, Decottignies P, van Tilbeurgh H, Myllykallio H, Liebl U (2006)
Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
J Biol Chem 281(33): 24048-24057  

9.  Urbonavicius J, Skouloubris S, Myllykallio H, Grosjean H (2005)
Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications.
Nucleic Acids Res 33(13): 3955-3964  

10.  Graziani S, Xia Y, Gurnon JR, Van Etten JL, Leduc D, Skouloubris S, Myllykallio H, Liebl U (2004)
Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella virus-1.  
J Biol Chem 279(52): 54340-54347  

11.  Leduc D, Graziani S, Lipowski G, Marchand C, Le Marechal P, Liebl U, Myllykallio H (2004)
Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers.
Proc Natl Acad Sci U S A 101(19): 7252-7257  

12.  Matsunaga F, Norais C, Forterre P, Myllykallio H (2003)
Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.
EMBO Rep 4(2): 154-158  

13.  Liebl U, Bouzhir-Sima L, Kiger L, Marden MC, Lambry JC, Negrerie M, Vos MH (2003)
Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli.  
Biochemistry 42(21): 6527-6535  

14.  Myllykallio H, Lipowski G, Leduc D, Filee J, Forterre P, Liebl U (2002)
 An alternative flavin-dependent mechanism for thymidylate synthesis.
Science 297(5578): 105-107 (highlighted in the same issue of Science).  

15.  Matsunaga F, Forterre P, Ishino Y, Myllykallio H (2001)
 In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin.
Proc Natl Acad Sci U S A 98(20): 11152-11157  

16.  Myllykallio H, Lopez P, Lopez-Garcia P, Heilig R, Saurin W, Zivanovic Y, Philippe H, Forterre P (2000)
Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon.
Science 288(5474): 2212-2215  

17.  Liebl U, Lipowski G, Negrerie M, Lambry JC, Martin JL, Vos MH (1999) Coherent reaction dynamics in a bacterial cytochrome c oxidase.
Nature 401(6749): 181-184