0.5X Tris-Borate-EDTA buffer. The gels were run at 80 V for 45 min, dried on Whatman DE81 paper, and visualized by a phosporimager (Typhoon 9210, Amersham). Data was analyzed using ImageQuant software (Amersham) and results were expressed as the ratio between the initial fraction of DNA bound to Topo III (f0) and the fraction of DNA bound to Topo III in the presence of the competitor (f) as a function of the competitor concentration in abscissa. Values correspond to the mean value of three independent experiments 6 SD. Cell culture The MRC5-V1/YFP-Topo III cell line is a stable clone of the ALT cell line MRC5-V1 transfected with YFP-Topo III [39] and was grown in Dulbecco’s modified Eagle’s medium (GIBCO/ Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen), 10 mM L-glutamine, and 400 mg/mL of geneticin. After transfection, cells were selected for 15 days with 400 mg/mL geneticin and cultures were sorted by FACS analysis and further grown in the presence of 400 mg/mL of geneticin. Antibodies Primary antibodies used in this work were raised against PML (PG-M3, mouse, sc-966 Santa Cruz Biotechnology), TRF2 (4A794, mouse, Upstate Biotechnology), BLM (C18, sc-7790 Santa Cruz Biotechnology), b-actin (clone AC-15, mouse, Sigma), active caspase-3 (IMG-144, mouse, Imgenex), c-H2AX (mouse, Upstate Biotechnology), TRF1 (C19, goat, sc-1977 Santa Cruz Biotechnology), and Topo IIIa (clone D6, rabbit (Wu et al., 2000)). Secondary antibodies used for western experiments were goat anti- rabbit and anti-mouse HRP conjugates (Upstate Biotechnology) and donkey anti-goat HRP conjugates (Abcam). For immunoflu- orescence experiments, goat anti-mouse Alexa fluor 488 and 568 as well as a goat anti-rabbit Alexa fluor 568 (Invitrogen) were used. Immunoblotting was performed according to [41]. G-overhang hybridization assays The nondenaturing hybridization assay to detect the 39 telomeric G-overhang was performed as described previously using the (AATCCC)4 oligonucleotide [42]. Immunofluorescence Cells were plated in 6-well culture plates on glass coverslips. After drug treatment, cells were washed with 1X PBS (pH 7.2), fixed with 4% paraformaldehyde, and permeabilized with 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose and 0.5% v/v Triton X-100 for 15 min at room temperature. Cells were then washed twice with PBS (pH 7.2). Cells were blocked with 1% BSA in 1X PBS for 1 hour and incubated with primary antibodies for 1 hour to 2 hours at room temperature. After washing with 1X PBS proteins of interest were detected by incubation for 30 min with fluorescently label secondary antibod- ies, then washed with 1X PBS. The nuclear DNA was stained with 0.1 mg/mL DAPI in PBS (pH 7.2) for 4 min. Cells were mounted in Shandon Immu-Mount medium (Thermo Scientific). Samples were observed with a DMR Leica microscope and images were captured with a Cool Snap HQ camera (Roper Scientific) controlled by Metamorph software (Roper Scientific). Final images are composed of arithmetic stacks of 15–25 deconvolued images, each 0.2 mM in z-step. Stacks of 15–30 images (16 bit grayscale) were acquired with a z-step of 0.2 mm with low illumination intensity. For the quantification of co-localizations, 50 nuclei were analyzed and results represent the mean 6 SD relative to YFP- Topo III or TRF1 foci, as indicated. Telomestatin Uncaps Telomeres Results Human Topo III binds to G-quadruplex but prefers single- stranded structures Purified recombinant Topo III interacts with single-stranded DNA, including the telomeric G-overhang sequence [39]. Since telomeric G-overhang is prone to form G-quadruplexes, we have examined whether the presence of guanines repeats influences the DNA binding of Topo III. A direct binding assay for Topo III to the telomeric G-overhang or to a mutated sequence unable to form G-quadruplex indicated that Topo III has a slight preference for single-stranded DNA that cannot fold into a G-quadruplex (result not shown). To quantify differences in binding, we have performed competition experiments with radioactively labeled single-stranded oligonucleotides (21G, S310 and 24C), unlabeled DNA oligonucleotides able to form G-quadruplex (21G, Pu22myc) or unable to form G-quadruplex (21Gmu, Pu22mu) [43,44]. The sequences of oligonucleotides are given in the Materials and Methods section. As an example, Figure 1A presents the analysis of the competition for Topo III binding to oligonucleotide 24C with either the G-quadruplex forming Pu22myc or its mutant Pu22mu, which is unable to fold into G-quadruplex. A 3-fold higher concentration of Pu22myc than Pu22mu was needed to inhibit Topo III binding to single-stranded oligonucleotide 24C. Similar differences between G-quadruplex-forming and mutant oligonucleotides were obtained for Topo III binding to 21G and S310 oligonucleotides (Figure 1B). The IC50s for competition with oligonucleotides that cannot form G-quadruplex was 4.3 to 10.1- fold lower than for G-quadruplex forming oligonucleotides in Figure 3. Inhibition of Topo III binding to telomeric sequence by telomestatin is due to G-quadruplex formation. A, Bandshift assay of purified Topo III (50 nM) to [32P]-labeled 21G (20 nM) in the presence of the indicated telomestatin concentrations. B, Bandshift assay of purified Topo III (50 nM) to [32P]-labeled 21Gmu (20 nM) in the presence of the indicated telomestatin concentrations. Telomestatin had no effect on binding of Topo III to the telomeric mutant sequence 21Gmu, which cannot form a G-quadruplex. doi:10.1371/journal.pone.0006919.g003 PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e6919