CHARACTERIZATION OF MG2-DEPENDENT 3'-PROCESSING ACTIVITY FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE IN-VITRO - REAL-TIME KINETIC-STUDIES USING FLUORESCENCE RESONANCE ENERGY-TRANSFER()

Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes the integration of HIV-1 DNA into the host chromosome. In vitro reactions with endogenous viral DNA require Mg2+ as the metal cofactor, whereas in vitro studies performed with short oligonucleotide substrates util ize Mn2+. In this study, we report that the donor processing activity of HIV-1 IN alters depending on the structure and length of the oligon ucleotide substrates. Increases in the length of the substrate cause a lterations in the efficiency of Mg2+-dependent donor processing activi ty, thereby reconciling this discrepancy between the in vivo and in vi tro HIV-1 IN mediated reactions. We have also found that the 3'-proces sing activity of HIV-IN is responsible for cleaving the junction betwe en the viral and target sequences of the recombination intermediate. I ts mechanism differs from the previously described disintegration reac tion in that the donor strands are regenerated without a joining react ion of the target strands. Kinetic studies of 3'-processing activity s uggest that the k(cat) (0.24/h) is very low. This implies that HIV-1 I N remains as a complex with the processed DNA prior to the strand tran sfer reaction.