RESUMEN
Humans with inherited heterozygous BRCA2 mutations have an increased risk of developing cancer; however, what triggers carcinogenesis in these individuals is unclear. Tan et al. find that environmental and metabolic aldehydes pose a threat to these individuals by promoting degradation of wild-type BRCA2 protein, thereby predisposing them to genomic instability and perhaps to cancer.
Asunto(s)
Aldehídos , Mutación , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Genes BRCA1 , Inestabilidad Genómica , Mutación de Línea Germinal , Heterocigoto , HumanosRESUMEN
HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases.
Asunto(s)
Anemia de Fanconi/genética , Anemia de Fanconi/patología , Proteínas HSP90 de Choque Térmico/genética , Pliegue de Proteína , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/química , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Mutación Missense , Dominios y Motivos de Interacción de Proteínas , Estrés Fisiológico , Gemelos MonocigóticosRESUMEN
The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Proteínas de Ciclo Celular , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Anemia de Fanconi , Empalme del ARN , Factor de Empalme U2AF , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Reparación del ADN , Endodesoxirribonucleasas , Exones , 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 A de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Unión Proteica , Precursores del ARN/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Empalmosomas/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/metabolismo , Factor de Empalme U2AF/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
In this issue of Molecular Cell, Brunner et al.1 reveal that eliminating FANCD2 from stalled forks via FBXL12-mediated degradation enables cells to tolerate oncogene-induced replication stress, making FBXL12 a promising target for cancer treatment.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismoRESUMEN
CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392. Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity.
RESUMEN
Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.
Asunto(s)
Proteína BRCA2/metabolismo , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/química , Exodesoxirribonucleasas/metabolismo , Factores de Transcripción/química , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Reparación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Células HeLa , Humanos , Ácido Mevalónico , Ratones , Complejos Multiproteicos , Mutación , Unión Proteica , Conformación Proteica , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/metabolismo , Recombinación GenéticaRESUMEN
The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance.
Asunto(s)
Reparación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/fisiología , Anemia de Fanconi/genética , Animales , Daño del ADN , Replicación del ADN , HumanosRESUMEN
DNA double-strand breaks (DSBs) can arise from multiple sources, including exposure to ionizing radiation. The repair of DSBs involves both posttranslational modification of nucleosomes and concentration of DNA-repair proteins at the site of damage. Consequently, nucleosome packing and chromatin architecture surrounding the DSB may limit the ability of the DNA-damage response to access and repair the break. Here, we review early chromatin-based events that promote the formation of open, relaxed chromatin structures at DSBs and that allow the DNA-repair machinery to access the spatially confined region surrounding the DSB, thereby facilitating mammalian DSB repair.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Neoplasias/genéticaRESUMEN
Anti-cancer drugs targeting the DNA damage response (DDR) exploit genetic or functional defects in this pathway through synthetic lethal mechanisms. For example, defects in homologous recombination (HR) repair arise in cancer cells through inherited or acquired mutations in BRCA1, BRCA2, or other genes in the Fanconi anemia/BRCA pathway, and these tumors have been shown to be particularly sensitive to inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP). Recent work has identified additional genomic and functional assays of DNA repair that provide new predictive and pharmacodynamic biomarkers for these targeted therapies. Here, we examine the development of selective agents targeting DNA repair, including PARP inhibitors; inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM); and inhibitors of classical non-homologous end joining (cNHEJ) and alternative end joining (Alt EJ). We also review the biomarkers that guide the use of these agents and current clinical trials with these therapies.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Reparación del ADN/fisiología , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Biomarcadores Farmacológicos , Daño del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN/genética , Genes BRCA1/efectos de los fármacos , Recombinación Homóloga , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Pirazinas/farmacología , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/metabolismo , Pirazoles/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.
Asunto(s)
Proteína BRCA2/genética , Sistemas CRISPR-Cas , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Endonucleasas de ADN Solapado/genética , Genes Letales , Neoplasias/genética , Interferencia de ARN , Mutaciones Letales Sintéticas , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Muerte Celular , Línea Celular Tumoral , Daño del ADN , Reparación del ADN por Unión de Extremidades , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas , Endonucleasas de ADN Solapado/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Enzimas Multifuncionales , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genéticaRESUMEN
BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.
Asunto(s)
Proteína BRCA1/deficiencia , Neoplasias de la Mama/enzimología , Replicación del ADN , ADN de Neoplasias/biosíntesis , Proteasas Ubiquitina-Específicas/metabolismo , Neoplasias del Cuello Uterino/enzimología , Animales , Proteína BRCA1/genética , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , ADN de Neoplasias/genética , Resistencia a Medicamentos , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones Desnudos , Mutación , Desnaturalización de Ácido Nucleico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Replicación del ADN/fisiología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteína BRCA1 , Proteína BRCA2 , Línea Celular , Roturas del ADN de Doble Cadena , ADN Helicasas/fisiología , Reparación del ADN , Proteínas de Unión al ADN , Desoxirribonucleasas , Endodesoxirribonucleasas , Inestabilidad Genómica/fisiología , Recombinación Homóloga/genética , Humanos , Proteína Homóloga de MRE11/metabolismo , Unión ProteicaRESUMEN
Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
Asunto(s)
Inestabilidad de Microsatélites , Repeticiones de Microsatélite/genética , Neoplasias/genética , Mutaciones Letales Sintéticas/genética , Helicasa del Síndrome de Werner/genética , Apoptosis/genética , Sistemas CRISPR-Cas/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Humanos , Modelos Genéticos , Neoplasias/patología , Interferencia de ARN , Proteína p53 Supresora de Tumor/metabolismo , Helicasa del Síndrome de Werner/deficienciaRESUMEN
Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.
Asunto(s)
Adenosina Trifosfatasas , ADN Polimerasa theta , Neoplasias , Novobiocina , Humanos , Adenosina Trifosfatasas/metabolismo , Replicación del ADN , ADN de Cadena Simple , ADN Polimerasa Dirigida por ADN/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Novobiocina/farmacologíaRESUMEN
The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Unión de Extremidades , Recombinación Homóloga , ADNRESUMEN
Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.
Asunto(s)
Anemia de Fanconi , Ratones , Animales , Anemia de Fanconi/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Left unrepaired, DNA interstrand crosslinks represent impassable hurdles for DNA replication, and their removal is a complicated stepwise process involving a variety of enzymes. In a recent paper in Science, Knipscheer et al. (2009) demonstrate that the Fanconi Anemia protein FANCD2 promotes multiple steps of the crosslink repair process.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Animales , HumanosRESUMEN
Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
Asunto(s)
Proteína BRCA1/deficiencia , Dineínas Citoplasmáticas/metabolismo , ADN/metabolismo , Genes BRCA1 , Proteína Homóloga de MRE11/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Aberraciones Cromosómicas , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Edición Génica , Inestabilidad Genómica/efectos de los fármacos , Recombinación Homóloga/efectos de los fármacos , Humanos , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Platino (Metal)/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica , Reparación del ADN por Recombinación/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two ß-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center. Without increasing the substrate affinity of USP12, the two ß-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.