Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.
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.
Department of Anatomical, Forensic Medicine, and Pathological Studies, Graduate School of Medicine, Kyoto University, Kyoto City, Kyoto Prefecture, Japan. tsuruyam@kuhp.kyoto-u.ac.jp
Tatsuaki
T
Tsuruyama
Tonau
T
Nakai
Rei
R
Ohmori
Munetaka
M
Ozeki
Keiji
K
Tamaki
Kenichi
K
Yoshikawa
D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003956:Dialysis; D004586:Electrophoresis; D019427:HIV Integrase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid
D004270:DNA, Circular; D004279:DNA, Viral; D011994:Recombinant Proteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1