Fig. 5

TOP1 interacts with G4 LTR III and this interaction inhibits TOP1 DNA relaxation activity. A–C G4 DNA pull-down assays performed with biotinylated oligonucleotides covering PQS present in HIV-1 LTR or c-myc promoters (see Fig. 4B and Additional file 5: Table S1). These oligonucleotides, either folded into a G-quadruplex (G4 folded) or hybridized to their complementary strand (DS) were attached to streptavidin magnetic beads and incubated with recombinant human 6His-TOP1 enzyme (A) or Jurkat cellular extracts (B, C). After several washes with increased salinity buffers, retained TOP1 was quantified by SDS-PAGE and western blotting. All these experiments were repeated at least 3 times. D Kinetic analysis by SPR of LTR III DNAs binding to TOP1. G4 LTR III WT and Mut1 DNAs, prepared in the running buffer containing 50 mM potassium chloride, were injected at increasing concentrations (111, 333 and 1000 nM) over the protein immobilized by amine coupling. Six independent experiments were performed with DNA samples injected in duplicate. Red lines represent the recorded sensorgrams. E Effect of WT and mutated G4 LTR III on TOP1 catalytic activity. This activity was measured by a DNA relaxation assay performed with recombinant human TOP1 (70 nM), a supercoiled pBR322 plasmid (200 ng) and different concentrations of G4-folded LTR III oligonucleotides (2.2 to 9 μM, from right to left). At the end of the reaction, the Open Circular (OC), Relaxed (R) and Supercoiled (SC) forms of the plasmid were separated by electrophoretic migration on a 1% Agarose gel and stained with Ethidium Bromide