Kinetic study of the HIV-1 DNA 3'-end processing

The FEBS Journal
Maksim SmolovEric Deprez

Abstract

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of prod...Continue Reading

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Citations

Jun 26, 2008·The Journal of Biological Chemistry·Ira B DickerMark R Krystal
Sep 6, 2011·Nucleosides, Nucleotides & Nucleic Acids·Sergey KorolevJulia Agapkina
Nov 11, 2009·Antimicrobial Agents and Chemotherapy·Olivier DelelisJean-François Mouscadet
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Nov 22, 2014·The Journal of Antimicrobial Chemotherapy·Soundasse MunirOlivier Delelis
Nov 18, 2008·Methods : a Companion to Methods in Enzymology·George MerkelAnna Marie Skalka
Jun 16, 2006·The Journal of Biological Chemistry·Elvire GuiotEric Deprez

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