Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA.
The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.
Agapkina
Julia
J
Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia and LBPA, UMR 8113 CNRS, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan Cedex, France.
Smolov
Maksim
M
Barbe
Sophie
S
Zubin
Evgenii
E
Zatsepin
Timofei
T
Deprez
Eric
E
Le Bret
Marc
M
Mouscadet
Jean-François
JF
Gottikh
Marina
M
D020029:Base Pairing; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D019427:HIV Integrase / Q000502:physiology*; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D013379:Substrate Specificity; D016662:Virus Integration / Q000502:physiology
D003432:Cross-Linking Reagents; D017931:DNA Primers; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D019427:HIV Integrase