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J. Biol. Chem., Vol. 283, Issue 41, 27838-27849, October 10, 2008

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Insight into the Integrase-DNA Recognition Mechanism

A SPECIFIC DNA-BINDING MODE REVEALED BY AN ENZYMATICALLY LABELED INTEGRASE^*

From the Laboratoire de Biologie et Pharmacologie Appliquée, CNRS, Ecole Normale Superieure Cachan, Institut d'Alembert, 61 Ave. du Président Wilson, 94235 Cachan, France

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 µM. 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.

Received for publication, April 29, 2008 , and in revised form, July 10, 2008.

^* This work was supported by TrioH European Project Grant FP6 503480, Agence Nationale de la Recherche Grant 06-PCVI-0015, and grants from CNRS and Institut d'Alembert. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both of these authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 33-147-40-23-94; Fax: 33-147-40-76-84; E-mail: deprez{at}lbpa.ens-cachan.fr.

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