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J. Biol. Chem., Vol. 281, Issue 32, 22707-22719, August 11, 2006

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Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity^*

Elvire Guiot, Kevin Carayon, Olivier Delelis, Françoise Simon, Patrick Tauc, Evgenii Zubin, Marina Gottikh, Jean-François Mouscadet, Jean-Claude Brochon, and Eric Deprez¹

From the Laboratoire de Biotechnologie et Pharmacologie Genetique Appliquee, CNRS, UMR8113, Ecole Normale Supérieure de Cachan, 61 av du Président Wilson, 94235 Cachan, France and the Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN·DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Received for publication, March 8, 2006 , and in revised form, May 24, 2006.

^* This work was supported by grants from the TrioH European Project (FP6 Grant 503480), the CNRS, the French National Agency for Research against AIDS, and the Russian Foundation for Basic Research (Grants 04-04-22000 and 05-04-48743). 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 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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