Asunto(s)
Médula Ósea , Rotura Cromosómica , Células Precursoras Eritroides , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Anemia de Fanconi , Mutación , Médula Ósea/metabolismo , Médula Ósea/patología , Preescolar , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patología , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Humanos , MasculinoRESUMEN
Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg-/- embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg-/- PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg-/- embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg-/- PGCs.
Asunto(s)
Anemia de Fanconi , Animales , Movimiento Celular/genética , Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Células Germinativas/metabolismo , Gónadas/metabolismo , Ratones , Transducción de SeñalRESUMEN
Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Regulación de la Expresión Génica , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN , Reparación del ADN , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Células HeLa , Humanos , Fosforilación , UbiquitinaciónRESUMEN
Fanconi anemia (FA) is a genetically and clinically heterogeneous disorder that predisposes patients to bone marrow failure (BMF), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). To study which genetic and phenotypic factors predict clinical outcomes for Japanese FA patients, we examined the FA genes, bone marrow karyotype, and aldehyde dehydrogenase-2 (ALDH2) genotype; variants of which are associated with accelerated progression of BMF in FA. In 88 patients, we found morphologic MDS/AML in 33 patients, including refractory cytopenia in 16, refractory anemia with excess blasts (RAEB) in 7, and AML in 10. The major mutated FA genes observed in this study were FANCA (n = 52) and FANCG (n = 23). The distribution of the ALDH2 variant alleles did not differ significantly between patients with mutations in FANCA and FANCG. However, patients with FANCG mutations had inferior BMF-free survival and received hematopoietic stem cell transplantation (HSCT) at a younger age than those with FANCA mutations. In FANCA, patients with the c.2546delC mutation (n = 24) related to poorer MDS/AML-free survival and a younger age at HSCT than those without this mutation. All patients with RAEB/AML had an abnormal karyotype and poorer prognosis after HSCT; specifically, the presence of a structurally complex karyotype with a monosomy (n = 6) was associated with dismal prognosis. In conclusion, the best practice for a clinician may be to integrate the morphological, cytogenetic, and genetic data to optimize HSCT timing in Japanese FA patients.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/genética , Secuencia de Bases , Anemia de Fanconi/genética , Anemia de Fanconi/mortalidad , Genotipo , Eliminación de Secuencia , Factores de Edad , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Alelos , Aloinjertos , Pueblo Asiatico , Supervivencia sin Enfermedad , Anemia de Fanconi/enzimología , Anemia de Fanconi/terapia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Femenino , Frecuencia de los Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Japón , Masculino , Tasa de SupervivenciaRESUMEN
Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.
Asunto(s)
Desarrollo Embrionario , Anemia de Fanconi/patología , Células Madre Hematopoyéticas/patología , Animales , Apoptosis , Ciclo Celular , Daño del ADN , Embrión de Mamíferos/patología , Eritrocitos/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Femenino , Ontología de Genes , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Hígado/embriología , Hígado/metabolismo , Ratones Endogámicos C57BL , Fenotipo , Placenta/metabolismo , Embarazo , Transcriptoma/genéticaRESUMEN
FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Reparación de la Incompatibilidad de ADN , ADN/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Reparación del ADN por Recombinación , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Línea Celular Tumoral , Clonación Molecular , ADN/metabolismo , Roturas del ADN de Doble Cadena , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Mariposas Nocturnas , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.
Asunto(s)
Reparación del ADN/genética , Reparación del ADN/fisiología , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Inestabilidad Cromosómica , Daño del ADN , Anemia de Fanconi/terapia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/deficiencia , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Técnicas de Inactivación de Genes , Terapia Genética , Histonas/metabolismo , Humanos , Mutación , Recombinasa Rad51/metabolismo , Proteasas Ubiquitina-Específicas/deficiencia , UbiquitinaciónRESUMEN
Transforming growth factor beta (TGF-ß) secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-ß in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-ß isoforms. In ten FA patients, mutations were detected in FANCA (n = 7), FANCG (n = 1) and FANCD2 (n = 2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. ß-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-ß1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-ß was absent in FA-D2 BM-MSCs. Absence of TGF-ß secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-ß1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-ß1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.
Asunto(s)
Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Análisis Mutacional de ADN , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Humanos , Mutación/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.
Asunto(s)
Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitinación , Línea Celular , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Transfección , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.
Asunto(s)
Curcumina/química , Proteína del Grupo de Complementación G de la Anemia de Fanconi/química , Anemia de Fanconi/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Biología Computacional , Proteínas de Unión al ADN , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/antagonistas & inhibidores , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ligandos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Biología de SistemasRESUMEN
Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Daño del ADN , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
DNA interstrand crosslinks (ICLs) are extremely deleterious lesions that are repaired by homologous recombination (HR) through coordination of Fanconi anemia (FA) proteins and breast cancer susceptibility gene 1 (BRCA1) product, but the exact role these proteins have remains unclear. Here we report that FANCG was modified by the addition of lysine63-linked polyubiquitin chains (K63Ub) in response to DNA damage. We show that FANCG K63Ub was dispensable for monoubiquitination of FANCD2, but was required for FANCG to interact with the Rap80-BRCA1 (receptor-associated protein 80-BRCA1) complex for subsequent modulation of HR repair of ICLs induced by mitomycin C. Mutation of three lysine residues within FANCG to arginine (K182, K258 and K347, 3KR) reduced FANCG K63Ub modification, as well as its interaction with the Rap80-BRCA1 complex, and therefore impeded HR repair. In addition, we demonstrated that K63Ub-modified FANCG was deubiquitinated by BRCC36 complex in vitro and in vivo. Inhibition of BRCC36 resulted in increased K63Ub modification of FANCG. Taken together, our results identify a new role of FANCG in HR repair of ICL through K63Ub-mediated interaction with the Rap80-BRCA1 complex.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Reparación del ADN por Recombinación , Ubiquitinación/fisiología , Secuencia de Aminoácidos , Intercambio Genético , Proteínas de Unión al ADN , Proteína del Grupo de Complementación G de la Anemia de Fanconi/química , Células HEK293 , Células HeLa , Chaperonas de Histonas , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Unión ProteicaRESUMEN
Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.
Asunto(s)
Linfocitos B/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Mutación Puntual , Hipermutación Somática de Inmunoglobulina/genética , Animales , Linfocitos B/inmunología , Secuencia de Bases , Western Blotting , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Humanos , Región de Cambio de la Inmunoglobulina/genética , Ratones de la Cepa 129 , Ratones Noqueados , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS/metabolismo , Reacción en Cadena de la Polimerasa , Recombinación GenéticaRESUMEN
Previous investigations have shown that areca nut extracts (ANE) or arecoline (ARE) causes DNA damage, which in turn contributes to oral cell carcinogenesis. To understand the role of microRNA (miRNA) in ANE-associated carcinogenesis, miRNA expression profile was examined in ANE-treated normal human oral fibroblasts. Among the miRNAs changed by ANE exposure, we found that ANE-induced miR-23a overexpression was correlated with an increase of γ-H2AX, a DNA damage marker. In addition, DNA double-strand breaks (DSB) repair that was determined by an in vivo plasmid-based assay was reduced in ANE-treated or miR-23a-overexpressed cells, suggesting the role of miR-23a in DSB repair. FANCG is one of Fanconi anemia susceptibility genes that participate in DSB repair pathway to prevent chromosomal aberrations. FANCG was predicted as a candidate target of miR-23a by TargetScan algorithm. This was confirmed by ectopic overexpression or knockdown of miR-23a. The correlation between miR-23a overexpression and areca nut-chewing habit could also be found in oral cancer patients. Finally, we showed that ANE-induced/ARE-induced miRNAs were significantly associated with the functional categories of "genetic disorders" and "cancer" using network-based analyses. In conclusion, our data showed for the first time that ANE-induced miR-23a was correlated with a reduced FANCG expression and DSB repair, which might contribute to ANE-associated human malignancies.
Asunto(s)
Areca/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , MicroARNs/biosíntesis , Extractos Vegetales/toxicidad , Arecolina/toxicidad , Línea Celular Tumoral , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Encía/efectos de los fármacos , Encía/metabolismo , Neoplasias Gingivales/tratamiento farmacológico , Neoplasias Gingivales/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , MicroARNs/genéticaRESUMEN
Human cytomegalovirus (HCMV) selectively relocalizes many DNA repair proteins, thereby avoiding a potentially detrimental damage response. In the present study, we evaluated interactions between HCMV and the homology-directed repair (HDR) pathway. In permissive human foreskin fibroblasts, a fluorescence-based double-stranded break repair assay was used to determine that HCMV stimulated HDR. Repair of both stably integrated and extrachromosomal reporter substrates was observed to increase. HDR was also stimulated through individual expression of the viral immediate-early protein IE1-72, mimicking full virus infection. These experiments further demonstrate HCMV's role in modulating critical cellular processes during a permissive infection.
Asunto(s)
Citomegalovirus/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Fibroblastos/virología , Proteínas Inmediatas-Precoces/metabolismo , Línea Celular , Citomegalovirus/genética , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Recombinasa Rad51/metabolismo , Replicación ViralRESUMEN
There is evidence that Fanconi anemia (FA) proteins play an important role in the repair of DNA interstrand cross-links (ICLs), but the precise mechanism by which this occurs is not clear. One of the critical steps in the ICL repair process involves unhooking of the cross-link from DNA by incisions on one strand on either side of the ICL and its subsequent removal. The ERCC1-XPF endonuclease is involved in this unhooking step and in the removal of the cross-link. We have previously shown that several of the FA proteins are needed to produce incisions created by ERCC1-XPF at sites of ICLs. To more clearly establish a link between FA proteins and the incision step(s) mediated by ERCC1-XPF, we undertook yeast two-hybrid analysis to determine whether FANCA, FANCC, FANCF, and FANCG directly interact with ERCC1 and XPF and, if so, to determine the sites of interaction. One of these FA proteins, FANCG, was found to have a strong affinity for ERCC1 and a moderate affinity for XPF. FANCG has been shown to contain seven tetratricopeptide repeat (TPR) motifs, which are motifs that mediate protein-protein interactions. Mapping the sites of interaction of FANCG with ERCC1, using site-directed mutagenesis, demonstrated that TPRs 1, 3, 5, and 6 are needed for binding of FANCG to ERCC1. ERCC1, in turn, was shown to interact with FANCG via its central domain, which is different from the region of ERCC1 that binds to XPF. This binding between FANCG and the ERCC1-XPF endonuclease, combined with our previous studies which show that FANCG is involved in the incision step mediated by ERCC1-XPF, establishes a link between an FA protein and the critical unhooking step of the ICL repair process.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/fisiología , Sitios de Unión , Reparación del ADN , Humanos , Unión Proteica , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Elaborate processes act at the DNA replication fork to minimize the generation of chromatid discontinuity when lesions are encountered. To prevent collapse of stalled replication forks, mutagenic translesion synthesis (TLS) polymerases are recruited temporarily to bypass DNA lesions. When a replication-associated (one-ended) double-strand break occurs, homologous recombination repair (HRR) can restore chromatid continuity in what has traditionally been regarded as an "error-free" process. Our previous mutagenesis studies show an important role for HRR in preventing deletions and rearrangements that would otherwise result from error-prone nonhomologous end joining (NHEJ) after fork breakage. An analogous, but distinct, role in minimizing mutations is attributed to the proteins defective in the cancer predisposition disease Fanconi anemia (FA). Cells from FA patients and model systems show an increased proportion of gene-disrupting deletions at the hprt locus as well as decreased mutation rates in the hprt assay, suggesting a role for the FANC proteins in promoting TLS, HRR, and possibly also NHEJ. It remains unclear whether HRR, like the FANC pathway, impacts the rate of base substitution mutagenesis. Therefore, we measured, in isogenic rad51d and fancg CHO mutants, mutation rates at the Na(+)/K(+)-ATPase alpha-subunit (ATP1A1) locus using ouabain resistance, which specifically detects base substitution mutations. Surprisingly, we found that the spontaneous mutation rate was reduced approximately 2.5-fold in rad51d knockout cells, an even greater extent than observed in fancg cells, when compared with parental and isogenic gene-complemented control lines. A approximately 2-fold reduction in induced mutations in rad51d cells was seen after treatment with the DNA alkylating agent ethylnitrosurea while a lesser reduction occurred in fancg cells. Should the model ATP1A1 locus be representative of the genome, we conclude that at least 50% of base substitution mutations in this mammalian system arise through error-prone polymerase(s) acting during HRR-mediated restart of broken replication forks.
Asunto(s)
Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Mutagénesis , Recombinasa Rad51/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etilnitrosourea/toxicidad , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Prueba de Complementación Genética , Humanos , Mutación , Recombinasa Rad51/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación GenéticaRESUMEN
Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow failure and complex congenital anomalies. Although mutations in FA genes result in a characteristic phenotype in the hematopoietic stem/progenitor cells (HSPCs), little is known about the consequences of a nonfunctional FA pathway in other stem/progenitor cell compartments. Given the intense functional interactions between HSPCs and the mesenchymal microenvironment, we investigated the FA pathway on the cellular functions of murine mesenchymal stem/progenitor cells (MSPCs) and their interactions with HSPCs in vitro and in vivo. Here, we show that loss of the murine homologue of FANCG (Fancg) results in a defect in MSPC proliferation and in their ability to support the adhesion and engraftment of murine syngeneic HSPCs in vitro or in vivo. Transplantation of wild-type (WT) but not Fancg(-/-) MSPCs into the tibiae of Fancg(-/-) recipient mice enhances the HSPC engraftment kinetics, the BM cellularity, and the number of progenitors per tibia of WT HSPCs injected into lethally irradiated Fancg(-/-) recipients. Collectively, these data show that FA proteins are required in the BM microenvironment to maintain normal hematopoiesis and provide genetic and quantitative evidence that adoptive transfer of WT MSPCs enhances hematopoietic stem cell engraftment.
Asunto(s)
Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Apoptosis/fisiología , Médula Ósea/fisiología , Adhesión Celular/fisiología , Proliferación Celular , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones MutantesRESUMEN
The structural protein nonerythroid alpha spectrin (alphaIISp) plays a role in the repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), in which there is a defect in ability to repair such cross-links. We have proposed a model in which alphaIISp, whose stability is dependent on FA proteins, acts as a scaffold to aid in recruitment of repair proteins to sites of damage. In order to get a clearer understanding of the proposed role of FA proteins in maintaining stability of alphaIISp, yeast two-hybrid analysis was carried out to determine whether FA proteins directly interact with alphaIISp and, if so, to map the sites of interaction. Four overlapping regions of alphaIISp were constructed. FANCG interacted with one of these regions and specifically with the SH3 domain in this region of alphaIISp. The site of interaction in FANCG was mapped to a motif that binds to SH3 domains and contains a consensus sequence with preference for the SH3 domain of alphaIISp. This site of interaction was confirmed using site-directed mutagenesis. Two FA proteins that did not contain motifs that bind to SH3 domains, FANCC and FANCF, did not interact with the SH3 domain of alphaIISp. These results demonstrate that one of the FA proteins, FANCG, contains a motif that interacts directly with the SH3 domain of alphaIISp. We propose that this binding of FANCG to alphaIISp may be important for the stability of alphaIISp in cells and the role alphaIISp plays in the DNA repair process.
Asunto(s)
Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Espectrina/química , Espectrina/metabolismo , Dominios Homologos src/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Consenso , Escherichia coli/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Espectrina/genética , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
Defects in DNA repair pathways have been involved in collapse of early neurogenesis leading to brain development abnormalities and embryonic lethality. However, consequences of DNA repair defects in adult neural stem and progenitor cells and their potential contribution in ageing phenotype are poorly understood. The Fanconi anaemia (FA) pathway, which functions primarily as a DNA damage response system, has been examined in neural stem and progenitor cells during developmental and adult neurogenesis. We have shown that loss of fanca and fancg specifically provokes neural progenitor apoptosis during forebrain development, related to DNA repair defects, which persists in adulthood leading to depletion of the neural stem cell pool with ageing. In addition, neural stem cells from FA mice had a reduced capacity to self-renew in vitro. Here, we expand upon our recent work and give further data examining possible implication of oxidative stress. Therefore, FA phenotype might be interpreted as a premature ageing of stem cells, DNA damages being among the driving forces of ageing.