TAILIEUCHUNG - Báo cáo khoa học: Kinetic study of the HIV)1 DNA 3¢-end processing Single-turnover property of integrase

The 3¢-processing of viral DNA extremities is the first step in the integra-tion process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detectedin vitro under conditions of excess enzyme. Despite such experi-mental conditions, steady-state Michaelis–Menten formalism is often applied to calculate characteristic equilibrium⁄kinetic constants of IN. | ềFEBS Journal Kinetic study of the HIV-1 DNA 3 -end processing Single-turnover property of integrase Maksim Smolov1 Marina Gottikh1 Vadim Tashlitskii1 Sergei Korolev1 Ilya Demidyuk2 Jean-Claude Brochon3 Jean-Francois Mouscadet3 and Eric Deprez3 1 Belozersky Institute of Physico-ChemicalBiology Moscow State University Russia 2 Institute of Molecular Genetics Russian Academy of Science Moscow Russia 3 LBPA UMR 8113 CNRS IFR121 Ecole Normale Superieure de Cachan France Keywords 3 -processing fluorescence anisotropy integrase protein-DNA interactions singleturnover kinetics Correspondence E. Deprez LBPA UMR8113 CNRS IFR121 Ecole Normale Superieure de Cachan 61 avenue du President Wilson 94235 Cachan cedex France Fax 33 1 47 40 76 84 Tel 33 1 47 40 23 94 E-mail deprez@ Received 17 October 2005 revised 20 December 2005 accepted 16 January 2006 doi 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 singleturnover mode and is intrinsically very slow single-turnover rate constant 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. .

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• molecular Biology
• tumor cells
• Antimicrobial
• medical knowledge
• medical research
• analysis of cytoplasmic
• oxidation
• Crystal structure
• bacteria
• hemoglobin
• medicine
• cell proliferation
• breast cancer
• chemical reaction
• gastric cancer
• hormone medicine
• biochemical studies
• environmental medicine