Fig. 3

Strand transfer activity in vitro. a Diagram of gel-based strand transfer assay. A pre-processed double-stranded oligo containing a 5′ fluorescent label is integrated into a supercoiled target plasmid (pUC19) in the presence of integrase and cofactor (Mg²⁺ or Mn²⁺). Single strand integration results in the formation of a tagged circle product accompanied by the relaxation of supercoiling. Concerted integration results in the formation of a 2 LTR coupled product, accompanied by the linearization of the plasmid. Reaction products are separated by agarose gel electrophoresis. b Example gels of IN 1F and IN MF strand transfer activity. The slowest-migrating band represents single strand integration events (tagged circle) with the linearized concerted integration product (2 LTR coupled) migrating further. The supercoiled target plasmid is visible in the ethidium bromide stain as the furthest-migrating band. Unintegrated fluorescent oligo is observed at the bottom of the gel. c Quantification of IN 1F and IN GSH strand transfer activity. Single strand integration events are shown in the top panels and concerted integration events are shown in the bottom panels. IN 1F shows greater concerted integration activity than IN GSH in the presence of either Mg²⁺ or Mn²⁺. d Quantification of IN 1F and IN MF strand transfer activity. Single strand integration events are shown in the top panels and concerted integration events are shown in the bottom panels. IN 1F shows greater concerted integration activity than IN MF in the presence of either Mg²⁺ or Mn²⁺. e Inhibition of strand transfer activity by raltegravir. Raltegravir more potently inhibits both the single strand (left) and concerted integration (right) activity of IN GSH and IN MF as compared to IN 1F. Data before normalization are plotted in Additional file 5: Figure S5. Data are plotted as mean ± SD of 3 replicates. * Denotes P < 0.05, ** denotes P < 0.01, and **** denotes P < 0.0001