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 previous ly by time-resolved fluorescence, using tryptophan anisotropy decay. This a pproach provides information on the size of macromolecules via the determin ation of rotational correlation times (theta). We have shown that, at submi cromolar concentration, IN is characterized by a long rotational correlatio n time (theta (20 degreesC) = 90-100 ns) corresponding to a high-order olig omeric form, likely a tetramer. In the present work, we investigated the se lf-assembly properties of the DNA-bound IN by using three independent fluor ophores. 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 degree sC) values of 15.5-19.5 and 23-27 ns, calculated from experiments performed at 25 degreesC and 37 degreesC, 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 degreesC) values were significantly higher when monitored at 37 degreesC a s compared with 25 degreesC. These results indicate that upon binding the v iral DNA end, the multimeric enzyme undergoes a dissociation, most likely i nto a homogenous monomeric form at 25 degreesC and into a monomer-dimer equ ilibrium at 37 degreesC.