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J. Biol. Chem., Vol. 281, Issue 17, 11530-11540, April 28, 2006

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Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA^*

Julia Agapkina, Maksim Smolov, Sophie Barbe, Evgenii Zubin, Timofei Zatsepin, Eric Deprez, Marc Le Bret, Jean-François Mouscadet¹, and Marina Gottikh

From the 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

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.

Received for publication, November 15, 2005 , and in revised form, February 21, 2006.

^* This work was supported by the TRIoH European Project FP6 Grant 503480, the Russian Foundation for Basic Research Grants 04-04-22000 and 05-04-48743, the CNRS (PICS27), and the French National Agency for Research on AIDS (ANRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Tables S1 and S2 and Fig. S1.

¹ To whom correspondence should be addressed: LBPA-CNRS UMR8113, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan Cedex, France. Tel.: 33-1-47-40-76-75; Fax: 33-1-47-40-76-84; E-mail: mouscadet{at}lbpa.ens-cachan.fr.

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