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1.
Stem Cells ; 28(7): 1186-95, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20506303

ABSTRACT

Fanconi anemia (FA) is a human genetic disease characterized by a DNA repair defect and progressive bone marrow failure. Central events in the FA pathway are the monoubiquitination of the Fancd2 protein and the removal of ubiquitin by the deubiquitinating enzyme, Usp1. Here, we have investigated the role of Fancd2 and Usp1 in the maintenance and function of murine hematopoietic stem cells (HSCs). Bone marrow from Fancd2-/- mice and Usp1-/- mice exhibited marked hematopoietic defects. A decreased frequency of the HSC populations including Lin-Sca-1+Kit+ cells and cells enriched for dormant HSCs expressing signaling lymphocyte activation molecule (SLAM) markers, was observed in the bone marrow of Fancd2-deficient mice. In addition, bone marrow from Fancd2-/- mice contained significantly reduced frequencies of late-developing cobblestone area-forming cell activity in vitro compared to the bone marrow from wild-type mice. Furthermore, Fancd2-deficient and Usp1-deficient bone marrow had defective long-term in vivo repopulating ability. Collectively, our data reveal novel functions of Fancd2 and Usp1 in maintaining the bone marrow HSC compartment and suggest that FA pathway disruption may account for bone marrow failure in FA patients.


Subject(s)
Endopeptidases/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Hematopoietic Stem Cells/metabolism , Animals , Arabidopsis Proteins , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Line , Cell Survival , Endopeptidases/deficiency , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Fanconi Anemia Complementation Group D2 Protein/deficiency , Hematopoietic Stem Cells/pathology , Mice , Mice, Inbred C57BL , Organ Specificity , Ubiquitin-Specific Proteases
2.
Mol Cell Biol ; 29(24): 6427-37, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19805513

ABSTRACT

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.


Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia/metabolism , Animals , Cell Line , Chromatin/metabolism , Cricetinae , Cricetulus , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/genetics , Endonucleases/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Gene Deletion , Humans , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Ubiquitin/metabolism
3.
J Clin Invest ; 117(5): 1440-9, 2007 May.
Article in English | MEDLINE | ID: mdl-17431503

ABSTRACT

The Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway-deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg(-/-)Atm(-/-) mice were found to be nonviable. Abrogation of ATM function in FA pathway-deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G- (FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway-deficient human cancers.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Fanconi Anemia Complementation Group C Protein/physiology , Fanconi Anemia Complementation Group G Protein/deficiency , Fanconi Anemia Complementation Group G Protein/genetics , Fanconi Anemia/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Signal Transduction/genetics , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Line, Transformed , Cell Line, Tumor , Cells, Cultured , DNA Damage , DNA-Binding Proteins/deficiency , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group C Protein/deficiency , Fanconi Anemia Complementation Group C Protein/genetics , Fanconi Anemia Complementation Group G Protein/physiology , HeLa Cells , Humans , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/deficiency , Tumor Suppressor Proteins/deficiency
4.
Mol Cell Biol ; 27(8): 3098-108, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17296736

ABSTRACT

The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.


Subject(s)
BRCA1 Protein/metabolism , Fanconi Anemia Complementation Group E Protein/metabolism , Fanconi Anemia/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Cell Cycle/drug effects , Cell Death/drug effects , Checkpoint Kinase 1 , Conserved Sequence , DNA Damage , DNA Replication/drug effects , Drug Resistance/drug effects , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group E Protein/chemistry , HeLa Cells , Humans , Mitomycin/pharmacology , Molecular Sequence Data , Mutant Proteins/metabolism , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , RNA, Small Interfering/metabolism , Ubiquitin/metabolism
5.
J Biol Chem ; 282(3): 2047-55, 2007 Jan 19.
Article in English | MEDLINE | ID: mdl-17082180

ABSTRACT

Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.


Subject(s)
Fanconi Anemia Complementation Group F Protein/chemistry , Mitomycin/pharmacology , Amino Acid Sequence , DNA Damage , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Signal Transduction , Transcription Factors/metabolism , Ubiquitin/chemistry
6.
J Biol Chem ; 281(16): 10896-905, 2006 Apr 21.
Article in English | MEDLINE | ID: mdl-16474167

ABSTRACT

Fanconi anemia (FA) is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. Eight of the 11 cloned Fanconi anemia gene products (FANCA, -B, -C, -E, -F, -G, -L, and -M) form a multisubunit nuclear complex (FA core complex) required for monoubiquitination of a downstream FA protein, FANCD2. FANCL, which possesses three WD40 repeats and a plant homeodomain (PHD), is the putative E3 ubiquitin ligase subunit of the FA complex. Here, we demonstrate that the WD40 repeats of FANCL are required for interaction with other subunits of the FA complex. The PHD is dispensable for this interaction, although it is required for FANCD2 mono-ubiquitination. The PHD of FANCL also shares sequence similarity to the canonical RING finger of c-CBL, including a conserved tryptophan required for E2 binding by c-CBL. Mutation of this tryptophan in the FANCL PHD significantly impairs in vivo mono-ubiquitination of FANCD2 and in vitro auto-ubiquitination activity, and partially impairs restoration of mitomycin C resistance. We propose a model in which FANCL, via its WD40 region, binds the FA complex and, via its PHD, recruits an as-yet-unidentified E2 for mono-ubiquitination of FANCD2.


Subject(s)
Fanconi Anemia Complementation Group L Protein/chemistry , Fanconi Anemia/metabolism , Alkylating Agents/pharmacology , Amino Acid Sequence , Animals , Cell Line , Chromosome Aberrations , DNA/chemistry , Dose-Response Relationship, Drug , Genetic Complementation Test , Glutathione Transferase/metabolism , Humans , Immunoblotting , Immunoprecipitation , Lymphocytes/metabolism , Mitomycin/metabolism , Mitomycin/pharmacology , Models, Biological , Molecular Sequence Data , Mutagenesis , Mutagenesis, Site-Directed , Mutation , Plasmids/metabolism , Point Mutation , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/metabolism , Retroviridae/genetics , Sequence Homology, Amino Acid , Tissue Distribution , Tryptophan/chemistry , Ubiquitin/chemistry
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