Telomestatin Uncaps Telomeres APBs in ALT cells and interact with shelterin components [19–21]. BLM is known to interact with Topoisomerase IIIa (Topo III) and two other proteins with OB-fold domains, RMI1 and RMI2, to form a RTR complex (RecQ/Topo III/RMI) essential to maintenance of genome stability through its function in the resolution of recombination intermediates (for recent reviews see [22,23]). In this complex, Topo III and BLM cooperate to convert double Holliday junctions (DHJ) to decatenated products in vitro [24]. RMI1 guides the binding of Topo III [25–27] and RMI2 regulates other protein-protein interactions in the complex [28,29]. The Topo III/BLM complex may function as a repair complex in response to replication defects and may restart stalled replication forks [30–32]. Following camptothecin treatment, a phosphorylated form of BLM dissociates from the Topo III/BLM complex at its PML storage sites and accumulates with c-H2AX at damage sites during replication [33]. Topo III belongs to the Type IA DNA topoisomerase subfamily, which is conserved among different organisms; it removes highly negative supercoils where single-stranded DNA is exposed [34]. In vitro this access can be provided by altering the secondary structure of the substrate using hyper-negative supercoiling or by the addition of single-stranded binding protein (RPA, RMI1) to assist Topo III binding [27]. Structural studies of E. coli Topo III revealed that the protein first recognizes the presence of single- stranded DNA through a specific DNA binding region and then induces protein rearrangements that activate the tyrosine catalytic region [35]. This ensures the recognition of the correct type of DNA by the active site and explains the requirement for cofactors to generate single-stranded DNA. Due to the diversity of complex structures that may form at telomere ends during fork progression, there is a requirement for factors that modulate topology, t-loop formation and resolution [36,37]. Among these factors, Topo III is an essential telomeric- associated component in ALT cells [38,39]. Topo III interacts with telomeric DNA and forms a complex with TRF2 and BLM at telomeres in ALT cells [39]. Inhibition of Topo III expression by Figure 1. Topo III preferentially binds to single-stranded oligonucleotides when compared to G-quadruplex-forming sequences. A, Competition experiment for Topo III (50 nM) binding to [32P]-labeled 24C oligonucleotide (20 nM) in the presence of either unlabeled Pu22myc or Pu22mu (see sequences in Material and Methods) at the indicated concentrations (in mM). B, Quantification of the competition experiments for Topo III binding to [32P]-labeled S310 (top), 21G (middle), and 24C (bottom) oligonucleotides (at 20 nM) with increasing concentrations (1 to 1000 nM) of unlabeled 21G (closed circles), 21Gmu (open circles), Pu22myc (closed triangles), and Pu22mu (open triangles) oligonucleotides. Results correspond to the mean 6 SD of three determinations. f0 is the initial fraction of DNA bound to Topo III and f the fraction of DNA bound to Topo III at a determined concentration of the competitor. doi:10.1371/journal.pone.0006919.g001 PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e6919