title: Insight into the integrase-DNA recognition mechanism. A specific DNA-binding mode revealed by an enzymatically labeled integrase.
abstract: Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Delelis
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Kevin
authors.initials: K
authors.lastname: Carayon
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Elvire
authors.initials: E
authors.lastname: Guiot
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Leh
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tauc
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Brochon
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Mouscadet
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France. Electronic address: deprez@lbpa.ens-cachan.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Deprez
mesh_terms: D000818:Animals; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006168:Guinea Pigs; D019426:Integrases / Q000737:chemistry*; D008956:Models, Chemical; D009994:Osmolar Concentration; D053150:Simian foamy virus / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins; D004247:DNA; D019426:Integrases