Volume 19, Issue 6, Pages 841-847 (September 2005) A FancD2-Monoubiquitin Fusion Reveals Hidden Functions of Fanconi Anemia Core Complex in DNA Repair  Nobuko Matsushita, Hiroyuki Kitao, Masamichi Ishiai, Naoki Nagashima, Seiki Hirano, Katsuya Okawa, Tomohiko Ohta, David S. Yu, Peter J. McHugh, Ian D. Hickson, Ashok R. Venkitaraman, Hitoshi Kurumizaka, Minoru Takata  Molecular Cell  Volume 19, Issue 6, Pages 841-847 (September 2005) DOI: 10.1016/j.molcel.2005.08.018 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Complementation of fancd2 Cells by FancD2 Protein Carrying a Monoubiquitination Site Mutation Fused with Wild-Type or Mutated Ubiquitin (A) Schematic representation of expressed proteins. In D2KR-monoUb, all Lys residues in ubiquitin were changed to Arg (all KR), while Ile44 in ubiquitin was mutated to Ala in D2KR-Ub-I44A. (B) Chromatin targeting of the D2KR-ubiquitin fusions. Wild-type (wt) and fancd2 cells expressing the indicated proteins were treated with MMC (500 ng/ml, 6 hr) or left untreated and then fractionated. Each fraction was separated by SDS-PAGE, and Western blotting was carried out using anti-FancD2 or anti-histone H4 antibody. WCE, whole-cell extract prepared by SDS-PAGE sample buffer. D2, wild-type FancD2 protein. (C–E) Colony survival assay in the presence of cisplatin of wild-type (wt) and fancd2 cells expressing D2KR-Ub (C), D2KR-monoUb (D), or D2KR-Ub-I44A (E). The mean and standard deviation (SD) of measurements carried out in triplicate are shown. The experiments were repeated at least twice with identical results. Molecular Cell 2005 19, 841-847DOI: (10.1016/j.molcel.2005.08.018) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Expression of D2KR Protein Fused with a Ubiquitin Moiety in fancc, fancg, and fancl Cells (A) Chromatin targeting of D2KR-Ub fusions. Cells expressing the indicated proteins were treated as in Figure 1B and then fractionated. Each fraction was separated and blotted as in Figure 1B. In case of fancl cells, data using D2KR fused with wild-type ubiquitin (D2KR-Ub) are shown, since D2KR-Ub achieved higher expression levels than D2KR-monoUb in fancl cells. (B–D) Colony survival assays in the presence of cisplatin of fancc (B), fancg (C), or fancl (D) cells, each expressing the indicated proteins. The mean and SD of measurements carried out in triplicate are shown. The experiments were repeated at least twice with identical results. Molecular Cell 2005 19, 841-847DOI: (10.1016/j.molcel.2005.08.018) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Expression of D2KR Protein Fused with Histone H2B in fancd2, fancc, fancg, and fancl Cells (A) Schematic representation of D2KR-histone H2B-GFP and D2KR-H2B protein. (B) Chromatin targeting of H2B-fused D2KR protein. Cells expressing the indicated proteins were treated as in Figure 1B or left untreated and then fractionated. Each fraction was separated and blotted as in Figure 1B. (C–F) Colony survival assays in the presence of cisplatin of fancd2 (C), fancc (D), fancg (E), and fancl cells (F) expressing indicated proteins. The mean and SD of measurements carried out in triplicate are shown. The experiments were repeated at least twice with identical results. Molecular Cell 2005 19, 841-847DOI: (10.1016/j.molcel.2005.08.018) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Localization of the FA Core Complex Components (A) Chromatin localization of the GFP-tagged FancC, FancG, or FancL. Mutant DT40 cells complemented with corresponding GFP-tagged chicken cDNA were treated with MMC (500 ng/ml, 6 hr) or left untreated and then fractionated. GFP-tagged proteins were pulled down with anti-GFP-beads and separated by SDS-PAGE, and pulled down proteins were detected by Western blotting using anti-GFP antibody. Each pull-down was performed on fractions derived from an equal number of starting cells. The amount of GFP-FancC in the fancc/fancg double mutant background and the PHD domain mutant (C305A) GFP-FancL was also examined by cell fractionation. (B) Subnuclear focus formation of GFP-FancC in fancc or fancc/fancg background. DT40 mutants stably expressing GFP-FancC were treated with MMC (500 ng/ml, 6 hr) or left untreated. Cytospin slides were prepared, stained with anti-FancD2 antibody, and observed under laser-scanning confocal microscopy. The lower left panel shows enlarged views of a foci-positive cell. Molecular Cell 2005 19, 841-847DOI: (10.1016/j.molcel.2005.08.018) Copyright © 2005 Elsevier Inc. Terms and Conditions