Telomestatin Uncaps Telomeres Figure 11. Model for the action of telomestatin in ALT cells. Short telomeres to be elongated are present in the APB platform and capped with proteins from the shelterin complex (Sh), including TRF1, TRF2, TIN2, TPP1, hRAP1 and POT1. The APBs also contains PML protein and proteins from the RTR complex (RTR), including Topo III, BLM, RMI1 and RMI2. Other recombination factors are not represented for sake of clarity. Telomere elongation takes place after homologous recombination (strand invasion); a possible mechanism for the resolution of the double Holliday junction occurring at the end of the recombination process involves Topo III/BLM recruitment by TRF2 at telomeres. In the presence of telomestatin, G- quadruplex stabilization induces APB disruption, which is a phenocopy of the siRNA-mediated Topo III deficiency, together with Topo III, BLM and TRF2 depletion, exposure of telomere ends to DNA damage (uncapping) and G-overhang degradation. A magnification of the image from Figure 6 is presented to illustrate the modification of the colocalization between PML, Topo III and TRF2. doi:10.1371/journal.pone.0006919.g011 The mechanistic details of the telomestatin-induced APB disruption are unclear. The depletion of several components of the shelterin complex, including TRF1, TRF2, TIN2 and RAP1 was previously shown to reduce formation of APBs [55]. Because telomestatin impairs the binding of POT1 and Topo III to the telomeric G-strand in vitro, the simplest explanation is that telomestatin disrupts the interactions to telomeres of telomere- binding proteins, inhibiting correct assembly of proteins on the APB platform. Although TRF2 does not directly bind to the telomeric G-overhang, G-quadruplex stabilization may induce indirect structural alterations at telomere ends or during replication, leading to the release and depletion of TRF2. Several pieces of evidence indicate that TRF2 affects telomere structure and represses homologous recombination events at the t- loop [1]. The N-terminal domain of TRF2 was recently reported to introduce a torsional stress in telomeric DNA, to promote strand invasion into duplex DNA and to bind to Holliday Junction structures [37,57]. We have proposed that Topo III might play a general role in ALT telomere physiology by resolving TRF2- mediated topological intermediates to modulate the t-loop formation or to modulate recombination processes [39]. It is likely that Topo III, which is essentially a nicking-closing enzyme that recognizes a single-stranded DNA region, catalyzes the resolution of the D-loop formed at t-loop or the double-Holliday junctions formed during telomere recombination (Figure 11). The G-quadruplex stabilization by telomestatin may interfere with t-loop formation/dissolution or with Holliday junction resolution at telomere ends. In agreement, in vitro experiments indicated that telomestatin inhibited the strand invasion reaction of a G-strand oligonucleotide into a telomeric duplex suggesting that the ligand is able to block the t-loop formation (JF Riou, unpublished results). The specific difference between ALT cells and telomerase-positive cells is due to the respective presence or absence of APBs where recombination takes place. Therefore, the inhibition of the double-Holliday junction resolution by telomes- tatin might be more deleterious for telomere capping than the inhibition of the t-loop formation/resolution. However we cannot exclude the possibility that the resolution of t-loop formation in ALT cells represents a critical process. Our data provides a possible clue to the important effects of telomestatin at telomeres in PLoS ONE | www.plosone.org 12 September 2009 | Volume 4 | Issue 9 | e6919