DNA-induced Polymerization of HIV-1 Integrase Analyzed with Fluorescence Fluctuation Spectroscopy

• Published in: The Journal of biological chemistry 2002-10, Vol.277 (41), p.38045-38052
• Main Authors: Vercammen, Jo, Maertens, Goedele, Gerard, Melanie, De Clercq, Erik, Debyser, Zeger, Engelborghs, Yves
• Format: Article
• Language: English
• Subjects: Buffers; DNA - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fluorescent Dyes - metabolism; HIV Integrase - chemistry; HIV Integrase - genetics; HIV Integrase - metabolism; Humans; Macromolecular Substances; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Polymers - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rhodamines - metabolism; Spectrometry, Fluorescence - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M205842200

Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 μm−1 under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 ± 0.6 × 108m−1. Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.