Telomestatin Uncaps Telomeres these experiments (Figure 1B), indicating that Topo III prefers single-stranded oligonucleotides relative to G-quadruplex forming oligonucleotides. Telomestatin inhibits Topo III binding to the telomeric G-overhang We next determined whether ligands that stabilize G-quadruplex formation interfere with the binding of Topo III to the telomeric G- strand. Telomestatin was isolated from Streptomyces annulatus and potently and selectively binds to and stabilizes telomeric G- quadruplexes [12–14]. In these experiments, we used the Wt26 oligonucleotide previously shown to form a G-quadruplex [45]. In the presence of telomestatin, we observed a strong inhibition of Topo III-Wt26 complex as detected by bandshift experiments (Figure 2A). Slight inhibition was observed at 0.03 mM telomestatin and no complex was observed at 0.1 mM; the IC50 was 60 nM. In contrast, telomestatin (up to 10 mM) had no effect on Topo III binding to the single-stranded oligonucleotide S310 (Figure 2B). Telomestatin efficiently inhibited Topo III binding to G-quadruplex forming 21G (IC50 equal to 300 nM) but not to the mutant sequence 21Gmu (IC50 .10 mM) (Figure 3A and B). We obtained similar results using other G-quadruplex ligands such as the pyridine dicarboxamide derivative 360A and the steroid derivative funtu- mine guanylhydrazone [17,46] (results not shown). We concluded that the stabilization of the G-quadruplex structure by specific ligands such as telomestatin efficiently impaired Topo III binding. Figure 5. Telomestatin induces a delocalization of Topo III from PML and TRF2. Representative images are of untreated MRC5-V1/YFP- Topo III cells or cells treated for 48 h with 2 mM telomestatin. A, Topo III detected by fluorescence of YFP-tagged Topo III (green) and PML detected by immunofluorescence (red). Dapi staining of DNA is shown in blue. B, Topo III detected by fluorescence of YFP-tagged Topo III (green) and TRF2 detected by immunofluorescence (red). Dapi staining of DNA is shown in blue. The extent of co-localization of Topo III and PML and of Topo III and TRF2 is decreased by telomestatin treatment relative to that in untreated cells. doi:10.1371/journal.pone.0006919.g005 PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e6919