Analysis of the DNA substrate structure and number of the processing sites on the activities of HIV-1 integrase in vitro.
A series of DNA substrates were synthesized to analyze the 3'-processing, integration and disintegration reactions taking place concurrently on the same DNA molecules and to evaluate the potential effects of various structural modifications of these molecules on the activities of HIV-1 integrase (IN). Our results indicate that DNA substrates containing multiple recognition sites for IN can produce efficiently the three activities of the enzyme. The 3'-processing and disintegration sites are recognized and processed by IN, both reactions being carried out in a competitive manner by the enzyme on the same DNA molecule. The presence of the gaps and unpaired nucleotides in the region surrounding the disintegration site had major deleterious effects on enzymes disintegration activity. Analysis of a different conformation at the base of the DNA hairpin has revealed a significant improvement of IN disintegration activity in the presence of double-stranded DNA on the 3' side of the disintegration site, suggesting that this region plays an important role in the stability of the enzyme-substrate complex. Interestingly, the efficiency of disintegration was strongly diminished in the presence of an unpaired nucleotide located immediately at the 3' end of the cleavage site. Overall, our results underline the extreme sensitivity of the HIV-1 IN to its substrates structure and conformation, especially for its disintegration activity, and the considerable importance of the disintegration activity in the reactions carried out in vitro by the purified enzyme.
Sayasith
K
K
Armand-Frappier Institute, Virology Research Center, Laval, Quebec, Canada. sayasik@yahoo.ca
Sauvé
G
G
Yelle
J
J
D004247:DNA / Q000378:metabolism*; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
D009841:Oligonucleotides; D004247:DNA; D019427:HIV Integrase