The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIa to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells Nassima Temime-Smaali1, Lionel Guittat2, Assitan Sidibe2, Kazuo Shin-ya3, Chantal Trentesaux1,2, Jean-Franc¸ ois Riou1,2* 1 Laboratoire d’Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Universite´ de Reims Champagne-Ardenne, Reims, France, 2 Laboratoire de Re´gulations et Dynamique des Ge´nomes, INSERM U565, CNRS UMR7196, Muse´um National d’Histoire Naturelle USM503, CP26, Paris, France, 3 Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Biological Systems Control Team, Tokyo, Japan Abstract In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIa (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed. Citation: Temime-Smaali N, Guittat L, Sidibe A, Shin-ya K, Trentesaux C, et al. (2009) The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIa to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells. PLoS ONE 4(9): e6919. doi:10.1371/journal.pone.0006919 Editor: Alfred Lewin, University of Florida, United States of America Received April 28, 2009; Accepted July 31, 2009; Published September 9, 2009 Copyright: 2009 Temime-Smaali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by the ‘‘Ligue Nationale contre le Cancer, Equipe Labellise´e’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: riou@mnhn.fr Introduction In vertebrates, the telomeres that cap chromosome ends are composed of tandem repeats of the sequence d(TTAGGG)n with a 39 single-stranded extension (G-overhang) associated with six protein factors (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) that form a protecting complex (shelterin) essential for genome stability [1]. Telomeres shorten at each round of cell division and cellular mechanisms that counteract this loss confer indefinite proliferation potential characteristic of cancerous cells [2]. Two mechanisms have been reported in the maintenance of telomere length. The first requires a specialized enzyme, called telomerase, which is able to copy, as a reverse transcriptase, the short TTAGGG motif at the 39 end of telomeres; the activity of telomerase is tightly controlled by shelterin [2]. The second mechanism involves recombination between telomeres, a mechanism known as Alternative Lengthening of Telomeres (ALT) [3]. ALT cells are characterized by the absence of telomerase activity, heterogeneous telomere length and the presence of nuclear foci termed ALT-associated PML bodies (APBs) that contain telomeric DNA, telomeric proteins TRF1 and TRF2, and DNA recombination/repair proteins [3]. In vitro, DNA oligonucleotides composed of the G-overhang DNA sequence may adopt a four-stranded structure called a G- quadruplex. Both direct and indirect evidence suggests that G- quadruplexes are also present in eukaryotic cells and that formation of this structure must be tightly regulated to allow DNA replication and cell division (for a review, see [4]). The binding of specific ligands to the telomeric G-quadruplex is a strategy to alter telomere functions and to inhibit cell growth. A number of G-quadruplex ligands have been synthesized during the last decade [5]. These compounds were initially derived from DNA intercalators [6] and now present selective binding properties to G-quadruplexes relative to duplex or single-stranded DNA [5]. The initial therapeutic rationale for use of these compounds in treatment of cancer was that inhibition of telomerase activity would inhibit unchecked cell proliferation [7,8], but several pieces of evidence suggest that these ligands must be considered as telomere targeting agents rather than simple telomerase inhibitors [9–11]. The natural product telomestatin, is one of the most potent and selective G-quadruplex binding small molecules known [12–14]. Treatment with telomestatin impairs telomere replication and provokes the release of POT1 and TRF2 from telomeres, which results in loss of single- and double-stranded telomeric DNA and inhibits growth of tumor cells [15]. However, little is known about the effect of this compound or other G-quadruplex ligands on telomerase-negative ALT cell lines, except that telomestatin inhibits cell proliferation[8,13,16,17]. G-quadruplex ligands also inhibit in vitro the activity of RecQ helicases WRN and BLM [18]. These helicases are associated with PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e6919