Analysis of the DNA substrate structure and number of the processing siteson 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 structu ral modifications of these molecules on the activities of HIV-1 integrase ( IN), Our results indicate that DNA substrates containing multiple recogniti on 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 enzy me on the same DNA molecule, The presence of the gaps and unpaired nucleoti des in the region surrounding the disintegration site had major deleterious effects on enzymes disintegration activity. Analysis of a different confor mation at the base of the DNA hairpin has revealed a significant improvemen t of IN disintegration activity in the presence of double-stranded DNA on t he 3 ' side of the disintegration site, suggesting that this region plays a n important role in the stability of the enzyme-substrate complex, Interest ingly, the efficiency of disintegration was strongly diminished in the pres ence of an unpaired nucleotide located immediately at the 3 ' end of the cl eavage site. Overall, our results underline the extreme sensitivity of the HIV-1 IN to its substrates structure and conformation, especially for its d isintegration activity, and the considerable importance of the disintegrati on activity in the reactions carried out in vitro by the purified enzyme.