Gue´ ret1,2* Abstract Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIa in Centromere Disjunction before Anaphase Se´ bastien Rouzeau1,2, Fabrice P. Cordelie` res1,2,3, Ge´ raldine Buhagiar-Labarche` de1,2, Ilse Hurbain4,5,6, Rosine Onclercq-Delic1,2, Simon Gemble1,2, Laura Magnaghi-Jaulin7, Christian Jaulin7, Mounira Amor- 1 Institut Curie, Centre de Recherche, Centre Universitaire, Baˆt Orsay, France, 2 CNRS UMR3348, Stress Ge´notoxiques et Cancer, Orsay, France, 3 Institut Curie, Centre de Recherche, Plateforme d’Imagerie Cellulaire et Tissulaire IBiSA, Centre Universitaire, Baˆt Orsay, France, 4 Institut Curie, Centre de Recherche, Paris, France, 5 Structure et Compartimentation membranaire, CNRS UMR144, Paris, France, 6 Plateforme d’Imagerie Cellulaire et Tissulaire IBiSA, CNRS UMR 144, Paris, France, 7 Institut de Ge´ne´tique et De´veloppement, CNRS UMR 6290, Universite´ de Rennes 1, Rennes, France Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIa, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIa in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIa inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIa, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges. Citation: Rouzeau S, Cordelie`res FP, Buhagiar-Labarche`de G, Hurbain I, Onclercq-Delic R, et al. (2012) Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIa in Centromere Disjunction before Anaphase. PLoS ONE 7(4): e33905. doi:10.1371/journal.pone.0033905 Editor: Beth A. Sullivan, Duke University, United States of America Received November 4, 2011; Accepted February 19, 2012; Published April 26, 2012 Copyright: 2012 Rouzeau 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: This work was supported by grants from the Institut Curie (PIC), the Centre National de la Recherche Scientifique (CNRS), the Cance´ropoˆ le/Re´gion Ile- de-France, the Ligue contre le Cancer Grand-Ouest, the Association pour la Recherche sur le Cancer (1071), the Agence Nationale de la Recherche (EpiCentr) and by fellowships from the Ministe`re de´le´gue´ de l’Enseignement Supe´rieur et de la Recherche and from the Ligue Nationale contre le Cancer. 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: mounira.amor@curie.fr Introduction The centromere is a highly differentiated chromosomal structure consisting, in human cells, of a-satellite DNA repeats [1]. It plays an essential role in cell division, particularly in kinetochore assembly, and in ensuring the segregation of equal number of chromosomes to daughter cells during mitosis [2]. The maintenance of centromere stability is thus essential to prevent chromosomal instability and cancer development [3]. Our interest in centromere stability began with the unexpected discovery that a helicase-like protein, PICH (Plk1-interacting checkpoint ‘‘heli- case’’) and the Bloom syndrome helicase (BLM) colocalized to centromeric ultrathin DNA threads that could not be counter- stained with conventional DNA dyes or antibodies against histones [4,5]. These threads were found to be common in all cultured normal cells tested, and are therefore probably physiological structures. These PICH- and BLM-positive DNA threads, which are also called ultrafine anaphase bridges (UFBs), are thought to contain unresolved DNA catenations between the centromeres separating during anaphase or to originate from incompletely replicated DNA [4,5]. Almost all the UFBs detected in untreated cells are of centromeric origin and have been proposed to prevent reactivation of the spindle assembly checkpoint (SAC) during early anaphase, by maintaining tension across centromeres [4,6]. As cells progress through anaphase, UFBs become progressively longer and decrease in number. They are no longer detectable by telophase. The resolution of centromeric UFBs requires topo- PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e33905