A rapid and quantitative assay for inhibition of 3′ cleavage activity of HIV-1 integrase
Autor: | Naisheng Fan, Kenneth B. Rank, Satish K. Sharma |
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Rok vydání: | 1997 |
Předmět: |
HIV Integrase
Cleavage (embryo) Substrate Specificity law.invention Endonuclease law Virology Nucleotide HIV Integrase Inhibitors Pharmacology Gel electrophoresis chemistry.chemical_classification biology Nucleotides Oligonucleotide Hydrolysis Molecular biology Recombinant Proteins Long terminal repeat Integrase Kinetics Biochemistry chemistry biology.protein Recombinant DNA Quercetin |
Zdroj: | Antiviral Research. 36:27-33 |
ISSN: | 0166-3542 |
Popis: | The human immunodeficiency virus-1 (HIV-1) integrase catalyzes the specific removal of two nucleotides at either 3′ end of each long terminal repeat (LTR) sequence of the proviral DNA duplex. The most commonly used in vitro assays for integrase employ 5′ end 32P-labeled double-stranded oligonucleotides and the product of integrase-associated endonuclease activity is visualized by denaturing gel electrophoresis followed by autoradiography. We report here a simple assay system based upon the liberation of [35S]GT dinucleotide from the 3′ end of a double-stranded U5 LTR sequence derived from HIV-1. The uncleaved labeled substrate and the unlabeled large product are removed by adsorption to polyethyleneimine cellulose followed by centrifugation. The small labeled GT dinucleotide product released in the supernatant is quantitated in terms of counts released as a function of time. Since the method is rapid and quantitative, it should be useful in the kinetic evaluation of inhibitors of the 3′ cleavage activity of HIV-1 integrase. |
Databáze: | OpenAIRE |
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