Biochemistry and Biophysics of Macromolecules - CNRS URA2185  


  HEADDr GOPAUL Deshmukh N. / gopauld@pasteur.fr
  MEMBERSDr CHESNE-SECK Marie-Laure / NIETO Silvia


  Annual Report

Site specific recombination by the integron integrase.

The Integron integrases belong to the site specific recombinases from the tyrosine recombinase family. They possess a conserved set of active site residues (R-K-H-R-H/W-Y) involved in DNA cleavage, stabilisation of the phospho-tyrosine transition state intermediate and Holliday junction. The integrons are mobile elements that carry an integrase enzyme, an insertion site (attI) and several gene cassettes composed of an open reading frame and an attC site. The integron integrases catalyse the integration of cassettes by recombining the attC sites with the attI, whilst they disperse cassettes by recombining attC sites. They have been shown to preferentially recombine the bottom strand of the recombination site. However, the structural basis of integron integrases’ use of varying sequences and single stranded DNA was still a conundrum. We are involved in the characterization of structure of the integron integrase VchIntIa from Vibrio cholerae in complex with a target attC site reconstituted from a folded bottom strand (VCRbs), by X-ray crystallography. The structure was solved by using single wavelength anomalous dispersion, with data recorded at the ESRF. The crystal structure revealed a tetrameric arrangement of monomers on DNA, with two protein molecules bound to each DNA site. The VCRbs DNA included key base mismatches which orient the enzyme on its substrate through specific recognition of the G20” base with tryptophans and of the T12” base a set of histidines and a proline.

The structure explained the single cut mechanism through the particular scaffolding of the β4,5 hairpin in trans across the synapse This data consolidate the role of enzymes that use base recognition without performing chemistry on the base itself. Site directed mutagenesis of the protein as well as the DNA bases, followed by biochemical and in vivo tests agree with the structural information. We are now in the process of approaching the attI x attC complex by similar methods.

The ability of bacteria to recombine incoming gene cassettes based on structural recognition elements instead of sequence information per se greatly enhances their ability to adapt to changing environmental conditions. This study opens the field to finding more efficient ways of limiting antibiotic resistance acquisition and spread, as well as new molecular tools for manipulating DNA.

The lab has also several other protein/DNA projects including the PhoP/PhoR two component system and other pathogenic integrases.

gopaul.jpg

Figure. X-ray structure of the VchIntIa-VCRbs integron integrase –DNA complex. Attacking (green) and non-attacking (magenta) units.



  Publications

Publications 2006 of the unit on Pasteur's references database




Activity Reports 2006 - Institut Pasteur
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