Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase.
HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D64, D116 and E152 of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V151E152S153 region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3'-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V151E152S153 region involvement in the integration reaction is more important than for the 3'-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.
Calmels
C
C
UMR-5097, CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, and IFR 66 Pathologies Infectieuses et Cancers, Bordeaux, France.
de Soultrait
V Richard
VR
Caumont
A
A
Desjobert
C
C
Faure
A
A
Fournier
M
M
Tarrago-Litvak
L
L
Parissi
V
V
D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004720:Endonucleases / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010641:Phenotype; D015003:Yeasts / Q000166:cytology
D018698:Glutamic Acid; D004247:DNA; D019427:HIV Integrase; D004720:Endonucleases