Nucleophile selection for the endonuclease activities of human, ovine, andavian retroviral integrases

Retroviral integrases catalyze four endonuclease reactions (processing, joi ning, disintegration, and nonspecific alcoholysis) that differ in specifici ty for the attacking nucleophile and target DNA sites. To assess how the tw o substrates of this enzyme affect each other, we per formed quantitative a nalyses, in three retroviral systems, of the two reactions that use a varie ty of nucleophiles. The integrase proteins of human immunodeficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DN A and during nonspecific alcoholysis of nonviral DNA. Although exogenous al cohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA Moreover, different nucleophiles were pr eferred when the various integrases acted on different DNA targets. In cont rast, the nicking patterns were independent of whether integrase was cataly zing hydrolysis or alcoholysis and were not influenced by the particular ex ogenous alcohol. Thus, although the target DNA influenced the choice of nuc leophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step befo re catalysis and suggest that integrase does not reach an active conformati on until target DNA has bound to the enzyme.