DNA binding induces dissociation of the multimeric form of HIV-1 integrase: a time-resolved fluorescence anisotropy study.
Self-assembly of HIV-1 integrase (IN) in solution has been studied previously by time-resolved fluorescence, using tryptophan anisotropy decay. This approach provides information on the size of macromolecules via the determination of rotational correlation times (theta). We have shown that, at submicromolar concentration, IN is characterized by a long rotational correlation time (theta(20 degrees C) = 90-100 ns) corresponding to a high-order oligomeric form, likely a tetramer. In the present work, we investigated the self-assembly properties of the DNA-bound IN by using three independent fluorophores. Under enzymatic assay conditions (10(-7) M IN, 2 x 10(-8) M DNA), using either fluorescein-labeled or fluorescent guanosine analog-containing oligonucleotides that mimic a viral end long terminal repeat sequence, we found that the DNA-IN complex was characterized by shorter theta(20 degrees C) values of 15.5-19.5 and 23-27 ns, calculated from experiments performed at 25 degrees C and 37 degrees C, respectively. These results were confirmed by monitoring the Trp anisotropy decay as a function of the DNA substrate concentration: the theta of IN shifted from 90-100 ns to lower values (<30 ns) upon increasing the DNA concentration. Again, the normalized theta(20 degrees C) values were significantly higher when monitored at 37 degrees C as compared with 25 degrees C. These results indicate that upon binding the viral DNA end, the multimeric enzyme undergoes a dissociation, most likely into a homogeneous monomeric form at 25 degrees C and into a monomer-dimer equilibrium at 37 degrees C.
Deprez
E
E
Laboratoire de Biotechnologie et Pharmacogénétique Appliquée (Centre National de la Recherche Scientifique-UMR8532), ENS-Cachan, 61 Avenue du Président Wilson, 94235 Cachan, France.
Tauc
P
P
Leh
H
H
Mouscadet
J F
JF
Auclair
C
C
Hawkins
M E
ME
Brochon
J C
JC
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D014364:Tryptophan / Q000737:chemistry
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; D014364:Tryptophan; D019427:HIV Integrase