Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA

Abstract

It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

Figure 1. AFM observation of integrase-target DNA complexes.

(A, B) Target DNA x_ky_k bound to HIV-1 integrase forming a loop-like structure or (C) a figure-eight-like structure. (D) DNA showing no HIV-1 integrase binding, where the arrow indicates unbound integrase. (E) Control experiment using x′_ky′_k including a ScaI digestion site AGTGACT. Bars indicate scale.

Figure 2. Number of integrase molecules bound in target DNA and modified DNA complexes.

Mean number of oligomeric integrases binding to the target DNA or modified target DNA strands (per one hundred strands) (mean ± s.d.). Results were determined from 5 independent experiments.

Figure 3. Distribution of the size of integrase-target DNA complexes.

(A) Line graphs showing representative measurement of the diameter across a twin-like structure of integrase bound to the DNA strands along the x- and y-axes. The upper and lower graphs show the longer and shorter diameter of the twin-globule structure individually. The photo in the inset is identical to the photo shown in Figure 1C. To the right are 3D crystal structures of integrase obtained from X-ray data (PDB 1K6Y) . Four integrase molecules associate in a tetrameric fashion in the DNA binding form. Monomers are represented by (a) green, (b) purple, (c) blue, and (d) orange. The diameter of the integrase dimer along the y-axis is approximately 5 nm, measured by the distance between two amino acids (E69 of c and I191 of d), represented in red. The thickness along the z-axis is approximately 3 nm, measured by the distance between Q62 of c and C65 of d, represented in red. These values were determined using Jmol. The scale bars are equivalent to approximately 1 nm. (B) Size of tetrameric integrase along the x-, y-, and z-axes (n = 123). (C) Histogram showing the distribution of the estimated volume (nm³) of the twin-like integrase structures bound to target DNA strands (n = 123).

Figure 4. Electrophoretic analysis of circular DNA bound to integrase.

(A) Electrophoretic analysis of control and target DNA following incubation with integrase. Graphs show electropherograms of signals corresponding to bands (i)–(iii). Peak area of individual signals (B) (i), (C)(ii), and (D)(iii) was determined using Multigauge software. Control lane depicts the electrophoresis of the random-sequence control DNA.