FANCM Connects the Genome Instability Disorders Bloom's Syndrome and Fanconi Anemia  Andrew J. Deans, Stephen C. West  Molecular Cell  Volume 36, Issue 6, Pages 943-953 (December 2009) DOI: 10.1016/j.molcel.2009.12.006 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Distinct Motifs in FANCM Promote Interactions with the FA and BS Complexes (A) Flag-affinity immunoprecipitations were carried out using extracts from 293 cells transfected with Flag-FANCM or the FANCM fragments indicated in (C). The indicated proteins were detected by western blotting. (B) Heat map of FANCM conservation among vertebrate homologs, performed using MUSCLE alignment and Jalview. The degree of conservation of individual residues is indicated by height (clustalX consensus) and color (BLOSUM62 score; yellow, high; brown, low). Black line indicates 30 residue moving average of consensus. (C) Flag-FANCM fragments used in (A) are indicated. The conserved MM1, MM2, and MM3 domains are boxed. (D) Schematic indicating the FANCM deletion mutants. (E) The Flag-FANCM-deletion mutants were immunoprecipitated as indicated in (A). (F) Extracts from 293 cells were incubated with biotinylated MM2 or control peptide TRF4 (BRCA2) or no peptide, and streptavidin pull-downs were performed. The input (lane A), flowthrough (lanes B–D), and elution (lanes E–G) are shown following western blotting with the indicated antibodies. See also Figure S1. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 FANCM Binds Components of the FA and Bloom's Complexes (A) 35S-labeled FANCA/B/C/E/F/G/L or BLM/RMI1/RMI2/TopoIIIα or luciferase (control) inputs were incubated with the indicated GST fusion bait protein, followed by glutathione pull-down and elution. The complexes were subjected to SDS-PAGE, and radiolabeled proteins were detected by autoradiography. (B) Schematic of the FANCF-Flag deletion mutants showing conserved regions and previously characterized interaction sites. The FANCM interaction site determined in (C) is shown. (C) Flag-affinity immunoprecipitation from 293 cells transfected with FANCF-Flag or the deletion mutants indicated in (B). Proteins were identified by western blotting. (D) Schematic of the Flag-RMI1 deletion mutants showing conserved regions and previously characterized interaction sites. The FANCM interaction site determined in (E) is shown. (E) Flag-affinity immunoprecipitation from 293 cells transfected with Flag-RMI1 or the fragments indicated in (D). See also Figure S2. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 FANCM Binds Independently to the FA and Bloom's Complexes and Provides a Link between the Complexes (A) FANCF-Flag was expressed in 293, control-siRNA, or FANCM-siRNA cells, followed by Flag pull-down and western blotting. (B and C) Immunoprecipitation with FANCM (M) or control antibodies (IgG) from wild-type, BS, or FA patient-derived extracts, followed by western blotting. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 The MM1 Motif Is Required for FANCD2 Ubiquitylation, Foci Formation, and MMC Resistance (A) Schematic diagram showing the constructs used to complement FANCM-depleted cells. The site of siRNA targeting and position of the wobble mutation (wob) in FANCM are indicated. (B) MMC survival curves of control, FANCM-siRNA knockdown, and complemented cell lines, as measured by the sulforhodamine B assay. Each point represents the average of three independent experiments plated in quadruplicate (± SD). (C) Control siRNA or stable FANCM-siRNA cells, or complemented cell lines, were either untreated or treated with 40 ng/ml MMC. After 7 hr growth, cells were harvested and extracts analyzed by western blotting for FANCD2 and FANCM. The ratio of FANCD2-L (ubiquitylated form) to FAND2-S (unmodified) is shown. Cells complemented with FANCM-ΔMM1, FANCM-ΔMM2, or FANCM-ΔMM1/2 or FANCM-ΔC are indicated. (D) Representative images of cells costained with anti-FANCD2 (red) and anti-γH2AX (green) antibodies, showing the formation of nuclear foci after damaging treatment. (E) Average values for the formation of FANCD2 foci in complemented cell lines 12 hr after treatment with 40 ng/ml MMC or 4 hr post 5 gray ionizing radiation. See also Figure S3. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 BLM Localization to Nuclear Foci Is FANCM Dependent after Treatment with Agents that Stall DNA Replication (A) Immunofluorescence staining for BLM foci in control siRNA (panels i–iv) and FANCM-siRNA stable cell lines (panels v–viii) after treatment with mitomycin C, camptothecin, or ionizing radiation. The percentages of foci scoring positive for more than five BLM foci/nucleus (out of a total of 200 scored nuclei) are indicated. (B) Dual immunofluorescent staining for BLM (green) and γH2AX (red). FANCM-siRNA cell lines were complemented with the indicated siRNA-resistant Flag-FANCM expression vectors. Scoring was carried out as in (A). Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Targeting of the FA and Bloom's Complexes by FANCM Is Required to Suppress SCE Formation (A and B) Representative mitotic spreads of MMC-treated 293 control siRNA or FANCM-siRNA cell lines stained to reveal SCEs (pink arrows). (C) SCEs were scored for 60 mitotic spreads for each cell line in the presence or absence of 10 ng/ml MMC. Each point represents the total number of SCEs in a single mitotic spread, and black bars indicate the average value for all spreads. ∗∗p > 0.001 compared to control siRNA, determined using the Mann-Whitney test. n.s., not significant. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Model for FANCM-Mediated Targeting of Multiple Repair Pathways to Sites of ICL Damage Translocation of FANCM along DNA or during fork reversal is promoted by the ATPase activity of the DEAH domain in the N terminus, which also interacts with HCLK2 and mediates ATR signaling and the cell-cycle checkpoint. Recognition of replication fork stalled at ICL damage occurs through the C-terminal domain of FANCM that requires dimerization with FAAP24. The FANCM-MM1 domain targets the FA core complex to chromatin through direct interaction with FANCF of the FA core complex, allowing FANCD2/I monoubiquitylation by FANCL. The MM2 domain targets the Bloom's complex to chromatin through interactions with RMI1 and TopoIIIα. The FA and Bloom's interactions, mediated by FANCM, are independent and result in an association of the two distinct repair complexes at sites of DNA damage. Molecular Cell 2009 36, 943-953DOI: (10.1016/j.molcel.2009.12.006) Copyright © 2009 Elsevier Inc. Terms and Conditions