RESUMO
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
Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.
Assuntos
Instabilidade Genômica , Mitógenos/fisiologia , Proteína do Retinoblastoma , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Transdução de Sinais/fisiologia , Aneuploidia , Animais , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Centrômero , Quebras de DNA de Cadeia Dupla , Variações do Número de Cópias de DNA , Fibroblastos/citologia , Camundongos , Mitógenos/farmacologia , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/deficiência , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/deficiência , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismoRESUMO
Fanconi anemia (FA) is a recessively inherited disease manifesting developmental abnormalities, bone marrow failure, and increased risk of malignancies. Whereas FA has been studied for nearly 90 years, only in the last 20 years have increasing numbers of genes been implicated in the pathogenesis associated with this genetic disease. To date, 19 genes have been identified that encode Fanconi anemia complementation group proteins, all of which are named or aliased, using the root symbol "FANC." Fanconi anemia subtype (FANC) proteins function in a common DNA repair pathway called "the FA pathway," which is essential for maintaining genomic integrity. The various FANC mutant proteins contribute to distinct steps associated with FA pathogenesis. Herein, we provide a review update of the 19 human FANC and their mouse orthologs, an evolutionary perspective on the FANC genes, and the functional significance of the FA DNA repair pathway in association with clinical disorders. This is an example of a set of genes--known to exist in vertebrates, invertebrates, plants, and yeast--that are grouped together on the basis of shared biochemical and physiological functions, rather than evolutionary phylogeny, and have been named on this basis by the HUGO Gene Nomenclature Committee (HGNC).
Assuntos
Medula Óssea/fisiopatologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Animais , Dano ao DNA/genética , Reparo do DNA/genética , Evolução Molecular , Anemia de Fanconi/metabolismo , Anemia de Fanconi/fisiopatologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismoRESUMO
OBJECTIVE: Oral squamous cell carcinoma (OSCC) may develop in young adults. In contrast to older patients, the well-known etiological factors, exposure to tobacco and alcohol, play a minor role in the carcinogenesis in this patient group. It has been suggested that an intrinsic susceptibility to environmental genotoxic exposures plays a role in the development of OSCC in these patients. The hypothesis was tested whether young OSCC patients have an increased sensitivity to induced chromosomal damage. SUBJECTS AND METHODS: Fourteen OSCC patients with an average age of 32 years (range 20-42) were selected. Peripheral blood lymphocytes and skin fibroblasts of patients and 14 healthy controls were subjected to the chromosome breakage test with Mitomycin C. This test is routinely used to identify Fanconi anemia patients, who are well-known for their inherited high sensitivity to this type of DNA damage, but also for the high risk to develop OSCC. Human papilloma virus status of the carcinomas was also determined. RESULTS: None of the 14 young patients with OSCC had an increased response in the MMC-chromosomal breakage test. All tumors tested negative for human papilloma virus. CONCLUSION: No evidence was obtained for the existence of a constitutional hypersensitivity to DNA chromosomal damage as a potential risk factor for OSCC in young adults.
Assuntos
Carcinoma de Células Escamosas/genética , Quebra Cromossômica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias Bucais/genética , Adulto , Carcinoma de Células Escamosas/sangue , Dano ao DNA , Anemia de Fanconi/genética , Feminino , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/sangue , Humanos , Masculino , Mitomicina/farmacologia , Neoplasias Bucais/sangue , Papillomaviridae , Fatores de Risco , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto JovemRESUMO
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
Roberts syndrome is an autosomal recessive disorder characterized by craniofacial anomalies, tetraphocomelia and loss of cohesion at heterochromatic regions of centromeres and the Y chromosome. We identified mutations in a new human gene, ESCO2, associated with Roberts syndrome in 15 kindreds. The ESCO2 protein product is a member of a conserved protein family that is required for the establishment of sister chromatid cohesion during S phase and has putative acetyltransferase activity.
Assuntos
Acetiltransferases/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico/fisiologia , Fenda Labial/genética , Fissura Palatina/genética , Ectromelia/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Acetiltransferases/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Ectromelia/metabolismo , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Linhagem , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
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
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 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
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/metabolismoAssuntos
Acetaldeído/toxicidade , Consumo de Bebidas Alcoólicas/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Etanol/toxicidade , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Acetaldeído/metabolismo , Aldeído Desidrogenase/deficiência , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/fisiopatologia , Dano ao DNA/genética , Reparo do DNA/genética , Etanol/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Transtornos do Espectro Alcoólico Fetal/etiologia , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/induzido quimicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , GravidezRESUMO
Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Proteínas Inibidoras de Apoptose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alcinos/farmacologia , Proteína 3 com Repetições IAP de Baculovírus/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Anemia de Fanconi/complicações , Anemia de Fanconi/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/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
Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.
Assuntos
Cisplatino/farmacologia , Genes BRCA1 , Genes BRCA2 , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Cisplatino/uso terapêutico , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação F da Anemia de Fanconi , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA/fisiologia , Feminino , Técnicas In Vitro , Mitomicina/farmacologia , Dados de Sequência Molecular , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
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.