Volume 25, Issue 3, Pages 331-343 (February 2007) Identification of FAAP24, a Fanconi Anemia Core Complex Protein that Interacts with FANCM  Alberto Ciccia, Chen Ling, Rachel Coulthard, Zhijiang Yan, Yutong Xue, Amom Ruhikanta Meetei, El Houari Laghmani, Hans Joenje, Neil McDonald, Johan P. de Winter, Weidong Wang, Stephen C. West  Molecular Cell  Volume 25, Issue 3, Pages 331-343 (February 2007) DOI: 10.1016/j.molcel.2007.01.003 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Interactions of FAAP24 with FANCM (A) The XPF family of flap/fork endonucleases. ERCC4 nuclease domains (red), HhH motifs (small blue boxes), and DEAH helicase domains (blue boxed DEAH) are indicated. Inactive ERCC4 and DEAH domains are indicated by the crossed domains. (B) Sequence alignment between the C terminus of H. sapiens FANCM (amino acids 1749–2048) and H. sapiens FAAP24. Identical and similar residues are indicated in filled and unfilled red boxes, respectively. Sequence alignments were carried out using ClustalW. (C) GSTFANCM1727–2048 and HISFAAP24 were coexpressed in E. coli. The GSTFANCM1727–2048/HISFAAP24 complex was then purified by GST affinity chromatography and applied to a gel filtration chromatography as described in Experimental Procedures. Fractions were analyzed by SDS-PAGE and the proteins visualized by Coomassie blue staining or western blotting by using SWE98 (FANCM) and SWE94 (FAAP24) antibodies. M, SDS-PAGE markers. The elution positions of gel filtration markers are indicated. (D) Purification of FANCM/FAAP24 complex. Baculovirus vectors carrying HISFANCMFLAG or HISFANCMFLAG and FAAP24 were expressed in insect cells, and extracts were subjected to chromatography by using anti-FLAG and nickel affinity columns. The eluates were analyzed by SDS-PAGE and visualized by silver staining (lanes b and c) or western blotting using SWE98 (FANCM) and SWE92 (FAAP24) antibodies. Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Specificity of the FANCM/FAAP24 Interaction (A) The indicated combinations of XPF family members were expressed in E. coli and analyzed by SDS-PAGE. The gel was then western blotted by using polyclonal antibodies against GST, EME2 (SWE57), FAAP24 (SWE92), or ERCC1 (FL-297) or monoclonal antibodies against EME1 (7H2). (B) Extracts from (A) were subjected to GST pull-downs, and the bead-bound complexes were detected by western blotting as described in (A). Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Interaction of FAAP24 with FANCM in the Mammalian Two-Hybrid Assay (A) Mammalian two-hybrid assay between FAAP24 and FA proteins. FAAP24, fused to the VP16 activation domain (AD), was cotransfected with FANCA, -B, -C, -D2, -E, -F, -G, -J, -L, or -M or FAAP100 linked to the GAL4 DNA binding domain (BD) to test for direct interactions in HEK293 cells. Luciferase induction is expressed relative to that measured when AD-FAAP24 was cotransfected with empty vector. Error bars show standard deviations for three independent experiments. (B) Mammalian two-hybrid assay between MUS81 and FAAP24 or EME1. Experiments were performed as described in (A). (C) Mammalian two-hybrid assay between FANCM and FAAP24 or EME1. Experiments were performed as described in (A). Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 In Vivo Interactions of FAAP24 with FANCM and the FA Core Complex (A) Schematic diagram indicating wild-type, FANCMdelC, and the K117R FANCM mutant. (B) FANCM was immunoprecipitated from nuclear extracts prepared from HEK293 cells stably expressing the FLAG-FANCM constructs shown in (A). Immunoprecipitation was carried out using anti-FLAG antibodies. The presence of FANCM and FAAP24 was detected by SDS-PAGE followed by western blotting. (C) FANCM immunocomplexes were isolated from HeLa nuclear extracts as described in Experimental Procedures, and constituent proteins were resolved by SDS-PAGE. The major bands were eluted from gel slices and the proteins identified by mass spectrometry. (D) FANCM, FAAP24, and FANCA antibodies were used to isolate immunocomplexes from HeLa nuclear extracts as described in Experimental Procedures. Proteins were analyzed by SDS-PAGE followed by western blotting for the indicated proteins. The load, immunoprecipitated (IP), and supernatant fractions are indicated. (E) Cytosolic (C) and nuclear extracts (NE) prepared from cell lines derived from a normal individual, an FA-M patient (EUFA867), and two FA-B patients (HSC230 and EUFA178) were analyzed for FANCM and FAAP24 by western blotting. BAF155, a component of the SWI/SNF chromatin-remodeling complex, and caspase-3 were included as loading controls for nuclear and cytosolic fractions, respectively. Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Cellular Effects of FAAP24 siRNA Depletion after ICL Damage (A) Inhibition of FAAP24 by siRNA reduces normal levels of FANCD2 ubiquitylation after DNA-damaging treatment. siRNA analyses were carried out as described in Experimental Procedures. Following mitomycin C (MMC) or cisplatin (CIS) treatment for 16 hr, FANCD2 and FAAP24 were detected by western blotting 72 hr post-siRNA treatment. The long and short isoforms of FANCD2 (FANCD2-L liter and FANCD2-S) are indicated. BAF155 was included as a loading control. (B and C) Clonogenic survival following treatment with DNA-damaging agents. HeLa cells were transfected with siRNAs as described in Experimental Procedures. At 70 hr posttransfection, cells were pulse treated with either cisplatin or mitomycin C for 3 hr and then grown in drug-free media until colonies were visible. FAAP24 protein levels were significantly depleted at 48–96 hr. Three independent experiments were performed, and in each experiment triplicate data points were obtained. Error bars show standard deviations for the experiment displayed in the figure. (D and E) Chromosomal aberrations following MMC treatment. HeLa cells were transfected with siRNAs according to the protocol described in (A). At 48 hr posttransfection, cells were treated with MMC (40 ng/ml) for 24 hr. At 72 hr, metaphase spreads were prepared and scored for chromosomal aberrations. See Table S1 for more experimental details and controls. Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 DNA Binding Activities of FAAP24 (A) Recombinant HISFAAP24 was expressed in E. coli and purified by Talon and SP Sepharose chromatography as described in Supplemental Experimental Procedures. Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. (B) Gel retardation assay for HISFAAP24. Reactions contained the indicated 32P-labeled substrates (approx 3 ng) and purified HISFAAP24 (2.5 μM). Reactions were incubated on ice for 30 min, and protein-DNA complexes were analyzed by 6% neutral PAGE followed by autoradiography. 32P labels are indicated with asterisks. Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 In Vitro Activities of the FANCM1727–2048/FAAP24 Complex (A) Recombinant FANCM1727–2048 and FANCM1727–2048/FAAP24 complex were expressed in E. coli and purified as described in Supplemental Experimental Procedures. Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. (B) Gel retardation assay for FANCM1727–2048 and FANCM1727–2048/FAAP24 complex. Reactions contained the indicated 32P-labeled substrates (approx 3 ng) and purified FANCM1727–2048 or FANCM1727–2048/FAAP24 (0.5 μM). (C) Comparison of the DNA binding activity of FANCM1727–2048/FAAP24 on ssDNA, splayed-arm, and 3′-flap substrates. Reactions were carried out as in (B) by using increasing amounts of FANCM1727–2048/FAAP24 (0.25 μM, lanes b, g, and l; 0.5 μM, lanes c, h, and m; 1 μM, lanes d, I, and n; and 2 μM, lanes e, j, and o). (D) Comparison of band-shift complexes formed by FANCM1727–2048, HISFAAP24, and FANCM1727–2048/FAAP24. The indicated proteins (0.5 μM) were incubated with the splayed-arm substrate as described in (B). (E) Analysis of XPF family members for 3′-flap endonuclease activity. Reactions contained 5′-32P-labeled DNA substrate and the indicated concentrations (indicated in nM) of purified MUS81/HISEME1, MUS81/HISEME2, or FANCM1727–2048/FAAP24. Reactions were incubated for 30 min at 37°C, and the products were deproteinized and then analyzed by neutral gel electrophoresis followed by autoradiography. 32P labels are indicated with asterisks. Molecular Cell 2007 25, 331-343DOI: (10.1016/j.molcel.2007.01.003) Copyright © 2007 Elsevier Inc. Terms and Conditions