FIG. 1.
Integrase assays. 32P-labeled (dark circles) oligonucleotides homologous to the HIV LTR incubated with recombinant HIV IN can be used to measure the catalytic activities of IN in vitro. In the absence of IN (−IN), no catalysis occurs. In the presence of IN (+IN), products are formed. Products can be separated from substrate by denaturing PAGE. (A) The 3′-end processing and strand transfer reaction. A 21-nucleotide oligomer is processed to the −2 product (19mer). In the same reaction, IN catalyzes the end-joining reaction, resulting in products larger than 21 nucleotides. A preprocessed 19mer can be used to uncouple the 3′-end processing activity from the strand transfer activity. (B) Disintegration assay. A 38-nucleotide substrate representing the partially integrated product can be resolved into its host and viral components. The resulting viral portion is 14 oligonucleotides in length. In both panels the reactions are performed in triplicate. The percent conversion of substrate to products can be quantified via phosphorimager analysis.