siRNA reduced ALT cell survival, but did not affect telomerase positive cell lines. Moreover, repression of Topo III expression in ALT cells induced telomere uncapping associated with reduced levels of TRF2 and BLM proteins [39]. This data suggested that the Topo III/BLM/TRF2 complex plays a role in the resolution of topological intermediates that arise during telomere recombination [39]. To examine whether Topo III binding at telomeric sequences and its localization at APBs is modulated by G-quadruplex formation, we have used the selective G-quadruplex ligand telomestatin. We found that Topo III binding is strongly inhibited by stabilization of G-quadruplex structures and that this ligand induces the disruption of APBs in ALT cells. This effect mimics the depletion of Topo III by RNA interference as it induces the depletion of the Topo III/BLM/TRF2 complex and results in telomere uncapping as evidenced by DNA damage at telomeres. We suggest that telomere maintenance in ALT cells may be particularly sensitive to G-quadruplex formation due to the need for the Topo III/BLM/TRF2 complex to resolve or modulate intermediate recombination/replication structures at telomeres. Materials and Methods Topo III and POT1 binding assays Topo IIIa was purified according to a previously published procedure [40]. Purified recombinant hPOT1, prepared after baculovirus expression, was a generous gift from Dr. D. Gomez Telomestatin Uncaps Telomeres (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). The oligonucleotides used in the study were: 21G: 59-GGGTTAGGGTTAGGGTTAGGG-39 21G mu3: 59-GGCTTACGGTTAGCGTTAGGG-39 Wt26: 59-TTAGGGTTAGGGTTAGGGTTAGGGTT-39 Pu22myc: 59-GAGGGTGGGGAGGGTGGGGAAG-39 Pu22mu: 59-GAGGGTGAAGAGGGTGGGGAAG-39 24C: 59-CCCTAACCCTAACCCTAACCCTAA-39 S310: 59-CCAGCTCTGCTTTGCATCTTT-39 Oligonucleotides were labelled at the 59 end with [c-32P]-ATP using T4 Polynucleotide Kinase (New England BioLabsH). Electrophoretic mobility shift assays using Topo III or hPOT1 were performed in 10 mL of the following solution: 50 mM HEPES (pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 4% w/v sucrose, 2% v/v glycerol, 0.1 mg/mL BSA, 0.02% w/v bromophenol blue, 20 nM labelled oligonucleotide and Topo III (50 nM) or hPOT1 (30 nM). For competition assays, the indicated concentrations of unlabelled oligonucleotides (1, 3, 10, 30, 100, 300, or 1000 nM) were added to the reaction mixture. For binding inhibition assays, the indicated concentrations of telomestatin were added in a volume of 1 mL. Telomestatin was prepared at 5 mM in 1:1 DMSO/MetOH and was dissolved to 100 mM in DMSO and further with water. The reaction mixture was incubated at room temperature for 30 min. Each individual mixture was separated immediately by electrophoresis on 1% agarose gels in Figure 2. Telomestatin inhibits Topo III and POT1 binding to telomeric G-overhangs. A, Bandshift assay using purified Topo III (50 nM) and [32P]-labeled Wt26 in the presence of the indicated concentrations of telomestatin. B, Bandshift assay using purified Topo III (50 nM) and [32P]- labeled S310 (20 nM) in the presence of the indicated concentrations of telomestatin. C, Bandshift assay using POT1 (30 nM) and [32P]-labeled Wt26 (20 nM) in the presence of the indicated concentrations of telomestatin. Telomestatin inhibits Topo III and POT1 binding to Wt26, which forms a G- quadruplex, but not to S310, which does not. doi:10.1371/journal.pone.0006919.g002 PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e6919