(Figure 5D). Thus, active Topo IIa is significantly less abundant at the centromeres of BLM-deficient cells and PICH-deficient cells than at those of control cells. These results also indicate that the helicase activity of BLM is involved in the recruitment of active Topo IIa to centromeres. Discussion We report here a new localization and function for BLM at centromeres. We also report that BLM cooperates with PICH at centromeres, and that both these proteins play an important role in a previously unknown mechanism involving the recruitment of active Topo IIa to centromeres to eliminate DNA catenation before the onset of anaphase. Our FISH experiments and EM images showed changes in the structure of centromeric chromatin in the absence of BLM or PICH (Figure 3). Recombinant PICH has recently been shown to have nucleosome remodeling activity in vitro, and BLM has been shown to be required for the chromatin remodeling function of PICH during anaphase in vivo [15]. On the other hand, changes in DNA topology are required for efficient mitotic decatenation by Top2, the fission yeast homolog of Topo IIa [19]. We therefore propose a model in which the combined action of BLM and PICH promotes the organization of centromeric chromatin, thereby rendering some centromeric catenates accessible to Topo IIa (Figure 6). This pathway may allow the Topo IIa-mediated resolution of some centromeric DNA catenations as soon as the cells enter prometaphase, thereby preventing centromeric non disjunction and the potential formation of additional UFBs that might interfere with abscission [7]. Our results also indicate that, in addition to their role in resolving physiological UFBs [5,15], BLM and PICH may participate in the prevention of UFB formation. Thus, the additional centromeric UFBs that form in BLM-deficient cells or in cells treated with inhibitors of the catalytic activity of Topo IIa may include both newly formed UFBs and unresolved physiolog- ical UFBs: these UFBs are probably similar in nature but of different origins. Indeed, cohesin complexes preserve catenation at centromeres until the onset of anaphase, accounting for the presence of physiological UFBs [7]. The resolution of centromeric catenation therefore depends on cohesin removal [7]. However, our results indicate that some centromeric catenations are processed before the onset of anaphase to prevent the formation of additional UFBs. Thus, physiological UFBs may arise from catenations locally protected by cohesin complexes and processed at anaphase onset, whereas additional UFBs may arise from catenations not resolved at an earlier step. In conclusion, the new centromeric decatenation mechanism reported here is probably the first step in a two-step centromeric decatenation process dependent on the coordinated action of BLM, PICH and Topo IIa. Moreover, our studies shed light on the mechanisms underlying the genetic instability and predispo- sition to cancer associated with Bloom’s syndrome. Materials and Methods Cell Lines, Cell Culture and Transfections The GFP-BLM construct was kindly provided by Nathan Ellis. The SV40-transformed fibroblast cell line GM08505B, derived from the BS patient 42 (RaFr) of the Bloom’s syndrome registry, was obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ, USA). BS and GFP-BLM cells were obtained by transfecting BS GM08505B cells with the EGFP-C1 vector alone (Clontech, Mountain View, CA), or with the same BLM and PICH in Centromere Disjunction vector containing the full-length BLM cDNA [20], respectively, in the presence of JetPEI reagent (Ozyme). GFP-I841T cells were obtained by transfecting BS GM08505B cells as described above, with the EGFP-C1 vector containing the full-length BLM cDNA mutated at codon 841 (see site-directed mutagenesis). HeLa and HeLa S3 cells, obtained from the American Tissue Culture Collection. HeLaV cells and HeLashBLM cells were obtained by trans- fecting cells with an empty pSM2 vector or with the same vector encoding a short hairpin RNA sequence directed against BLM (Open Biosystems, clone V2HS-89234), respectively, with JetPEI reagent. After 48 h, selection with 1 to 5 mg/ml puromycin (Invivogen) was applied. Individual colonies were isolated and cultured in medium containing 0.5 mg/ml puromycin [10,21]. All cell lines were cultured in DMEM (Gibco) supplemented with 10% decomplemented FCS (Invitrogen), L-glutamine and antibiotics. Cells were transfected with siRNAs directed against BLM (48 h), PICH (72 h) and Rad21 (72 h) (ON-TARGETplus, SMARTpool, Dharmacon) and with a control non-targeting pool of siRNA (ON-TARGETplus siCONTROL Non Targeting Pool, Dharmacon) (100 nM final concentration) in the presence of Dharmafect 1 (Dharmacon) according to the manufacturer’s instructions. The sequences of all siRNA oligonucleotides used are presented in Table S1. The pool of siRNAs targeting PICH has been shown to have no effect on nocodazole-induced SAC (spindle assembly checkpoint) activation [22]. Chemicals Etoposide (Sigma) was used at a final concentration of 0.5 mM. Plasmid Construction and Site-directed Mutagenesis Site-directed mutagenesis was performed with the EGFP-C1 vector containing the full-length BLM cDNA: Ile-841 and Gly-891 were mutated with the QuikChange XL site-directed mutagenesis kit (Agilent) according to the manufacturer’s instructions. Primer 1 (59-CCCAGGGTACAGAAGGACACCCTGACT- CAGCTGAAG - 39) and primer 2 (59-CTTCAGCTGAGT- CAGGGTGTCCTTCTGTACCCTGGG- 39) were used to mutate Ile-841 and primer 3 (59-GCACCACCCATATGATT- CAGAGATAATTTACTGCCT- 39) and primer 4 (59-AGG- CAGTAAATTATCTCTGAATCATATGGGTGGTG- 39) were used to mutate Gly-891. Western-blot Analysis Cells were lysed in 8 M urea buffer (8 M urea, 150 mM b- mercaptoethanol, and 50 mM Tris (pH 7.4) in water), sonicated and heated. Samples equivalent to 2.5 105 cells were subjected to electrophoresis in NuPAGE Novex 4–12% Bis-Tris precast gels (Invitrogen). The procedures used for immunoblotting have been described elsewhere [23]. Antibodies All the commercial antibodies were used according to the manufacturers’ specifications. The primary antibodies were used for immunofluorescence (IF), immunoprecipitation (IP) or immu- noblotting (IB) at the following concentrations: rabbit anti-BLM (1:5000 for IB and 1:200 for IF, AB476 from Abcam), goat anti- BLM (1:150 for IF, C18 from Santa-Cruz Biotechnology), CREST serum (1:500 for IF, from Antibody Incorporated (Fig. 1), kindly provided by I. Bahon-Riedinger (Fig. 2)), mouse anti-cyclin B1 (1:500 for IF, sc-245 from Santa Cruz), mouse anti-Rad21 (1:1000 for IB, 53A303 from Upstate), mouse anti-Topo II alpha (1:1000 PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e33905