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Article Dans Une Revue Journal of Biological Chemistry Année : 2006

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.

Résumé

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.

Domaines

Biophysique

Dates et versions

hal-00216092 , version 1 (24-01-2008)

Identifiants

Citer

Elvire Guiot, Kevin Carayon, Olivier Delelis, Françoise Simon, Patrick Tauc, et al.. Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.. Journal of Biological Chemistry, 2006, 281 (32), pp.22707-19. ⟨10.1074/jbc.M602198200⟩. ⟨hal-00216092⟩
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