Published OnlineFirst March 9, 2010; DOI: 10.1158/1541-7786.MCR-09-0534 Molecular DNA Damage and Cellular Stress Responses Cancer Research The Bloom Syndrome Protein Limits the Lethality Associated with RAD51 Deficiency Kenza Lahkim Bennani-Belhaj1,2, Sébastien Rouzeau1,2, Géraldine Buhagiar-Labarchède1,2, Pauline Chabosseau1,2, Rosine Onclercq-Delic1,2, Emilie Bayart1, Fabrice Cordelières3,4, Jérôme Couturier5,6, and Mounira Amor-Guéret1,2 Abstract Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the re- combinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family. Mol Cancer Res; 8(3); 385–94. ©2010 AACR. Introduction cently, SUMOylation of BLM has been shown to regulate its association with RAD51 and its function in HR-medi- Bloom's syndrome displays one of the strongest known ated repair of damaged replication forks (13). In several correlations between chromosomal instability and a high models, it has been proposed that BLM restarts replication risk of cancer at an early age. Bloom's syndrome is caused after the stalling of the fork, thus preventing HR-depen- by mutations in the BLM gene, which encodes BLM, a dent fork restart (14-16). The Bloom's syndrome pheno- RecQ 3′-5′ DNA helicase (1). The hallmark of Bloom's type also includes a significant increase in the frequency of syndrome cells is a high rate of sister chromatid exchange anaphase bridges and lagging chromosomes during cell di- (SCE), generally thought to be the consequence of replica- vision, indicating a defect in sister chromatid separation tion-dependent double-strand breaks. SCEs are mediated during mitosis (17). BLM localizes to anaphase bridges by homologous recombination (HR; refs. 2-4). BLM defi- and is required for their elimination (17), suggesting that ciency is associated with replication abnormalities and an BLM helps to resolve aberrant chromosome structures gen- increase in HR (1). In vitro, BLM unwinds DNA struc- erated during DNA replication (17, 18). Moreover, BLM tures mimicking replication forks and HR intermediates. is associated with 4′,6-diamidino-2-phenylindole (DAPI)– It catalyzes the regression of replication forks and works negative ultrafine DNA bridges (UFB), which are thought with topoisomerase IIIα, RMI1, and RMI2 to dissolve to be derived from replication or recombination intermedi- double Holliday junctions (5-10). BLM inhibits the D- ates (17). In particular, BLM localizes to UFBs and to con- loop formation catalyzed by RAD51 by displacing ventional DAPI-positive anaphase bridges associated with RAD51 from ssDNA, thereby disrupting nucleoprotein fi- fragile sites and is required for their resolution (19). laments (11). In vivo evidence that BLM can disrupt RAD51 downregulation significantly increases fragile-site RAD51 polymerization has also been reported (12). Re- expression under both normal conditions and replication stress (20). These data suggest that a functional relation- Authors' Affiliations: 1Institut Curie, Centre de Recherche, Centre ship between BLM and RAD51 may also exist during mi- Universitaire; 2CNRS, UMR 2027; 3Institut Curie, Centre de Recherche, Plateforme d'Imagerie Cellulaire et Tissulaire, Centre Universitaire; tosis. Using RNA interference to downregulate BLM and/ 4CNRS, UMR 146, Orsay, France; 5Institut Curie, Hôpital, Service de orRAD51inHeLacells,weshowherethatBLMand Génétique Oncologique; and 6INSERM, U830, Paris, France RAD51 interact functionally in both interphase and mito- Note: Supplementary data for this article are available at Molecular tic cells. Indeed, we report an epistatic interaction between Cancer Research Online (http://mcr.aacrjournals.org/). BLM and RAD51 in SCE formation. We also show that (a) Current address for E. Bayart: Centre de Génétique Moléculaire, UPR RAD51 acts upstream from BLM to prevent anaphase CNRS 2167, Bâtiment 24, Avenue de la Terrasse, Gif sur Yvette, France. bridge formation; (b) RAD51 downregulation is associated Corresponding Author: Mounira Amor-Guéret, Institut Curie, CNRS UMR 2027, Centre Universitaire, Bâtiment 110, Orsay 91405, France. with an increase in the frequency of BLM-positive ana- Phone: 33-1-69-86-30-53; Fax: 33-1-69-86-94-29. E-mail: mounira. phase bridges, but not of BLM-associated UFBs; and (c) [email protected] BLM downregulation increases the levels of cell death as- doi: 10.1158/1541-7786.MCR-09-0534 sociated with RAD51 downregulation. Our results indicate ©2010 American Association for Cancer Research. that BLM acts downstream from RAD51 to resolve www.aacrjournals.org 385 Downloaded from mcr.aacrjournals.org on October 3, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 9, 2010; DOI: 10.1158/1541-7786.MCR-09-0534 Lahkim Bennani-Belhaj et al. RAD51-mediated Holliday junctions or to rescue anaphase gel electrophoresis and immunoblotting were as previously bridges resulting from RAD51 deficiency, probably at dif- described (24). ficult-to-replicate DNA sequences such as fragile sites. Antibodies All the commercial antibodies were used according to the Materials and Methods manufacturers' specifications. The primary antibody used against BLM was ab476 (1:1,000; rabbit, Abcam). We used Cell Cultures and Transfections rabbit polyclonal antibodies against RAD51 (1:1,000; AB- HeLa cells were used, as previously described (21). He- 1, Calbiochem) and β-actin (1:10,000; Sigma). LaV cells and HeLashBLM cells were obtained by trans- Horseradish peroxidase–conjugated goat anti-mouse IgG fecting HeLa cells with an empty pSM2 vector or a and goat anti-rabbit IgG (Santa Cruz Biotechnology) were pSM2 vector encoding a short hairpin RNA (shRNA) se- used at a dilution of 1:5,000. Alexa Fluor-488–conjugated quence directed against BLM (Open Biosystems, clone donkey anti-goat IgG antiserum was obtained from Molec- V2HS-89234), respectively, using JetPEI reagent (Ozyme) ular Probes, Inc., and used at a dilution of 1:600. according to the manufacturer's instructions. After 48 h, selection with 1 to 5 μg/mL puromycin (Invivogen) was Reverse Transcription-PCR Analysis applied. Individual colonies were isolated and maintained Total RNA was extracted with Trizol reagent (Invitrogen). in growth medium containing 0.5 μg/mL puromycin. The first-strand cDNA was synthesized with 250 ng of ran- HeLa cells producing H2B fused to green fluorescent pro- dom hexamers (Invitrogen), 5 μg of RNA, and SuperScript tein (GFP) were kindly provided by Dr. Kevin F. Sullivan II reverse transcriptase (Invitrogen) in the presence of 40 (NUI Galway, Galway, Ireland; ref. 22). For siRNA tran- units of RNase Inhibitor (Promega). Amplification was car- sient transfection assays, 3 × 105 to4×105 cells were added ried out with Taq polymerase (Promega) and specific ssDNA to 3 mL of medium in each well of a six-well plate. Cells primers for BLM (5′-gaagatgctcaggaaagtgac-3′ and 5′-gcag- were transfected by incubation with siRNAs specific for tatgtttattctgatctttc-3′) and 18S rRNA (QuantumRNA 18S BLM or RAD51 (ON-TARGETplus, SMARTpool, Dhar- Internal Standards, Ambion) as an internal control for 25 macon) or with negative control siRNAs (ON-TARGET- PCR cycles (denaturation at 95°C for 40 s, annealing at plus siCONTROL Non Targeting Pool, Dharmacon) at a 56°C for 30 s, extension at 72°C for 40 s). final concentration of 100 nmol/L (single transfections) or 200 nmol/L (cotransfections) for 24 to 72 h, in the presence SCE Assays and Chromosomal Aberration Analysis of DharmaFECT I (Dharmacon), used according to the Cells were transfected as indicated. After 24 to 40 h of manufacturer's instructions. siRNA transfection, cells were transferred to slides and cul- tured in the presence of 10 μmol/L 5-bromodeoxyuridine Chemicals (Sigma) at 37°C in an atmosphere containing 5% CO2. After 40 h, colchicine (Sigma) was added to a final concen- Hydroxyurea (Sigma) was used at a final concentration μ of 2 mmol/L. tration of 0.1 g/mL and the cells were incubated for 1 h. Cells were incubated in hypotonic solution [1:6 (v/v) FCS- distilled water] and fixed with a 3:1 (v/v) mixture of meth- Assays to Assess the Efficiency of Colony Formation anol-acetic acid. Cells were then stained by incubation Untreated cells were plated in a drug-free medium at with 10 μg/mL Hoechst 33258 (Sigma) in distilled water three different densities, in triplicate, for the counting of for 20 min, rinsed with 2× SSC (Euromedex), exposed to 30 to 300 clones depending on expected survival. After UV light at 365 nm at a distance of 10 cm for 105 min, 14 to 21 d of incubation, colonies were fixed and stained rinsed in distilled water, stained by incubation with 2% with methylene blue (5 g/L in 50% water and 50% meth- Giemsa solution (VWR) for 16 min, rinsed in distilled wa- anol) and scored. Only experiments giving a linear correla- ter, dried, and mounted. Chromosomes were observed tion between the different dilutions were considered. with a Leica DMRB microscope at ×100 magnification. Colony-forming efficiency was estimated by dividing the Metaphases were captured with a SONY DXC 930 P cam- number of colony-forming units by the number of cells era and SCEs or chromosomal aberrations were analyzed. plated. Immunofluorescence Microscopy Western Blot Analysis Immunofluorescence staining was done as previously de- Cells were lysed by incubation in 1% SDS in water or in scribed (25).