RESUMO
The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single-molecule technique for visualizing encounters of replication forks with ICLs as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs and that these lesions are not always absolute blocks to replication.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/genética , DNA/biossíntese , Anemia de Fanconi/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Cricetinae , DNA Helicases/genética , Proteínas de Ligação a DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Lisina/química , Lisina/genética , Lisina/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Dobramento de Proteína , Multimerização Proteica , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , DNA/genética , Dano ao DNA , DNA Helicases/química , DNA Helicases/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Grupos de Complementação da Anemia de Fanconi , Humanos , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Troca de Cromátide IrmãRESUMO
Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remain unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status.
Assuntos
Proteína BRCA2/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/genética , Dano ao DNA , Replicação do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Reguladoras de Apoptose , Proteína BRCA2/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Ficusina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas Nucleares/genética , Plasmídeos/metabolismo , Recombinação Genética , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.
Assuntos
Proteínas de Ligação a DNA/genética , Desoxirribonucleases/genética , Anemia de Fanconi/genética , Predisposição Genética para Doença/genética , Fenótipo , Apoptose/genética , Apoptose/efeitos da radiação , Sequência de Bases , Exoma/genética , Anemia de Fanconi/patologia , Mutação em Linhagem Germinativa/genética , Humanos , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Análise de Sequência de DNA , Raios UltravioletaRESUMO
The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.
Assuntos
Proteína BRCA2/fisiologia , Neoplasias da Mama/genética , Anemia de Fanconi/genética , Proteínas Nucleares/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteína do Grupo de Complementação N da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Predisposição Genética para Doença , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The diagnosis of VACTERL syndrome can be elusive, especially in the prenatal life, due to the presence of malformations that overlap those present in other genetic conditions, including the Fanconi anemia (FA). We report on three VACTERL cases within two families, where the two who arrived to be born died shortly after birth due to severe organs' malformations. The suspicion of VACTERL association was based on prenatal ultrasound assessment and postnatal features. Subsequent chromosome breakage analysis suggested the diagnosis of FA. Finally, by next-generation sequencing based on the analysis of the exome in one family and of a panel of Fanconi genes in the second one, we identified novel FANCL truncating mutations in both families. We used ectopic expression of wild-type FANCL to functionally correct the cellular FA phenotype for both mutations. Our study emphasizes that the diagnosis of FA should be considered when VACTERL association is suspected. Furthermore, we show that loss-of-function mutations in FANCL result in a severe clinical phenotype characterized by early postnatal death.
Assuntos
Canal Anal/anormalidades , Esôfago/anormalidades , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/genética , Rim/anormalidades , Deformidades Congênitas dos Membros/diagnóstico , Deformidades Congênitas dos Membros/genética , Mutação , Fenótipo , Coluna Vertebral/anormalidades , Traqueia/anormalidades , Aborto Induzido , Quebra Cromossômica , Diagnóstico Diferencial , Exoma , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Nascido Vivo , Masculino , Gravidez , Diagnóstico Pré-Natal , Índice de Gravidade de DoençaRESUMO
Fanconi anemia (FA) is a rare recessive disorder with chromosomal instability, congenital abnormalities, and a high cancer risk. The breast cancer susceptibility gene BRCA2 (FANCD1) is one of the 16 genes involved in this recessive disease. We have identified a novel mutation of the splice donor site of intron 1 in the noncoding region of BRCA2 in a Japanese FA family. This mutation may account for the FA phenotype in a patient originally reported to have biallelic mutations in BRCA2. Subsequent functional studies revealed that one of the mutations, K2729N, was a neutral change. As reported here, a more careful analysis resulted in the identification of a novel splice site mutation. Functional analysis using a mouse embryonic stem cell-based assay revealed that it causes aberrant splicing, reduced transcript levels and hypersensitivity to DNA damaging agents, suggesting that it is likely to be pathogenic. Although similar pathogenic variants in the noncoding region of BRCA1 and 2 were not identified in a cohort of 752 familial breast cancer cases, we still think this finding is relevant for mutation analysis in Hereditary Breast and Ovarian Cancer Syndrome families in a diagnostic setting.
Assuntos
Proteína BRCA2/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Animais , Proteína BRCA1/genética , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Sítios de Splice de RNARESUMO
Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.
Assuntos
Archaea/química , DNA Helicases/genética , Reparo do DNA , Anemia de Fanconi/genética , Hemaglutininas Virais/genética , Ligases/genética , Proteínas Virais de Fusão/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Evolução Biológica , DNA/metabolismo , DNA Helicases/deficiência , DNA Helicases/metabolismo , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Humanos , Imunoprecipitação , Ligases/deficiência , Ligases/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Transporte Proteico , Ubiquitina/metabolismo , Proteínas Virais de Fusão/deficiênciaRESUMO
The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.
Assuntos
Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Mutação/genética , RNA Helicases/deficiência , RNA Helicases/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi , Teste de Complementação Genética , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Deleção de SequênciaRESUMO
Fanconi anemia (FA) is a rare genetic disorder characterized by congenital malformations, progressive bone marrow failure (BMF), and susceptibility to malignancies. FA is caused by biallelic or hemizygous mutations in one of 15 known FA genes, whose products are involved in the FA/BRCA DNA damage response pathway. Here, we report on a patient with previously unknown mutations of the most recently identified FA gene, SLX4/FANCP. Whole exome sequencing (WES) revealed a nonsense mutation and an unusual splice site mutation resulting in the partial replacement of exonic with intronic bases, thereby removing a nuclear localization signal. Immunoblotting detected no residual SLX4 protein, which was consistent with abrogated interactions with XPF/ERCC1 and MUS81/EME1. This cellular finding did not result in a more severe clinical phenotype than that of previously reported FA-P patients. Our study additionally exemplifies the versatility of WES for the detection of mutations in heterogenic disorders such as FA.
Assuntos
Exoma/genética , Anemia de Fanconi/genética , Mutação , Recombinases/genética , Sequência de Bases , Códon sem Sentido , Análise Mutacional de DNA , Anemia de Fanconi/metabolismo , Feminino , Humanos , Immunoblotting , Dados de Sequência Molecular , Sítios de Splice de RNA/genética , Recombinases/metabolismo , Adulto JovemRESUMO
SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers--BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2-negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C-induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.
Assuntos
Neoplasias da Mama Masculina/genética , Neoplasias da Mama/genética , Mutação , Recombinases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA , Saúde da Família , Anemia de Fanconi/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Sítios de Splice de RNA/genéticaRESUMO
Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5' single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3' single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2') substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2' substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.
Assuntos
RNA Helicases DEAD-box/química , DNA Helicases/química , Distúrbios no Reparo do DNA/enzimologia , DNA/química , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Distúrbios no Reparo do DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Deleção de Sequência , Especificidade por Substrato , SíndromeRESUMO
The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.
Assuntos
Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Quebra Cromossômica , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Mutação/genética , Troca de Cromátide Irmã/genética , Xeroderma Pigmentoso/genética , Adolescente , Sequência de Bases , Pré-Escolar , RNA Helicases DEAD-box/deficiência , DNA Helicases/deficiência , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Neoplasias/genética , Linhagem , Fenótipo , Polônia , Gravidez , SíndromeRESUMO
Fanconi anaemia (FA) is a rare recessive disorder marked by developmental abnormalities, bone marrow failure, and a high risk for the development of leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF, has also been documented in sporadic tumours in non-FA patients. To study whether there is a causal relationship between FA pathway defects and tumour development, we have generated a mouse model with a targeted disruption of the FA core complex gene Fancf. Fancf-deficient mouse embryonic fibroblasts displayed a phenotype typical for FA cells: they showed an aberrant response to DNA cross-linking agents as manifested by G(2) arrest, chromosomal aberrations, reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous mice were viable, born following a normal Mendelian distribution, and showed no growth retardation or developmental abnormalities. The gonads of Fancf mutant mice functioned abnormally, showing compromised follicle development and spermatogenesis as has been observed in other FA mouse models and in FA patients. In a cohort of Fancf-deficient mice, we observed decreased overall survival and increased tumour incidence. Notably, in seven female mice, six ovarian tumours developed: five granulosa cell tumours and one luteoma. One mouse had developed tumours in both ovaries. High-resolution array comparative genomic hybridization (aCGH) on these tumours suggests that the increased incidence of ovarian tumours correlates with the infertility in Fancf-deficient mice and the genomic instability characteristic of FA pathway deficiency.
Assuntos
Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Tumor de Células da Granulosa/genética , Luteoma/genética , Neoplasias Ovarianas/genética , Animais , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Proteína do Grupo de Complementação F da Anemia de Fanconi/deficiência , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.
Assuntos
Anemia de Fanconi/genética , Ligases/genética , Proteínas Nucleares/genética , Deleção de Sequência , Sequência de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA2/genética , Sequência de Bases , Aberrações Cromossômicas , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Humanos , Ligases/deficiência , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismoRESUMO
Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.
Assuntos
Cromossomos Humanos X , Anemia de Fanconi/genética , Metilação de DNA , Mecanismo Genético de Compensação de Dose , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Feminino , Teste de Complementação Genética , Ligação Genética , Humanos , Masculino , Mutação , Proteínas Nucleares/metabolismo , Linhagem , Receptores Androgênicos/metabolismoRESUMO
The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/deficiência , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Alelos , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovário/anormalidades , Ovário/metabolismo , Fenótipo , Troca de Cromátide Irmã , Taxa de Sobrevida , Testículo/anormalidades , Testículo/metabolismoRESUMO
FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Camptotecina/farmacologia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , DNA Helicases/deficiência , Resistência a Medicamentos/genética , Resistência a Medicamentos/fisiologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Expressão Gênica , Humanos , Mutação , Tolerância a Radiação/genética , Tolerância a Radiação/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Ubiquitinação/genética , Raios UltravioletaRESUMO
The capacity to maintain genomic integrity is shared by all living organisms. Multiple pathways are distinguished that safeguard genomic stability, most of which have originated in primitive life forms. In human individuals, defects in these pathways are typically associated with cancer proneness. The Fanconi anemia pathway, one of these pathways, has evolved relatively late during evolution and exists - in its fully developed form - only in vertebrates. This pathway, in which thus far 13 distinct proteins have been shown to participate, appears essential for error-free DNA replication. Inactivating mutations in the corresponding genes underlie the recessive disease Fanconi anemia (FA). In the last decade the genetic basis of this disorder has been uncovered by a variety of approaches, including complementation cloning, genetic linkage analysis and protein association studies. Here we review these approaches, introduce the encoded proteins, and discuss their possible role in ensuring genomic integrity.