RESUMEN
Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.
Asunto(s)
Anemia de Fanconi , Humanos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Lisina/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Ubiquitinación , ADN/metabolismo , Daño del ADN , Reparación del ADNRESUMEN
BACKGROUND: Ovarian cancer (OVCA) has unique epigenetic alterations and defects in homologous recombination (HR). Despite initial sensitivity to platinum-based chemotherapy, HR dysfunctional tumors eventually acquire drug resistance. Fanconi anemia (FA) is characterized by bone marrow failure (BMF) and a reduced ability to eradicate DNA interstrand cross-links (ICL). However, the mechanism of chemoresistance mediated by FANCI was unclear in OVCA. OBJECTIVE: We explore to identify whether FANCI was involved in chemoresistance in OVCA. METHODS: FANCI expression and epigenetic alterations were analyzed, respectively, using TIMER and cBioPortal. The correlation between FANCI expression and the survival of OVCA patients was analyzed using Kaplan-Meier Plotter, GSE63885, and TCGA-OVCA dataset. FANCI expression in OVCA was detected by immunohistochemistry. Cell proliferation, migration, and invasion in FANCI inhibiting cells were assessed by CCK-8 and Transwell. Apoptosis and DNA damage were examined by flow cytometry and immunofluorescence. Meanwhile, the activity of caspase 3/7 was detected by Caspase-Glo® 3/7 kit. In addition, the expression of FANCI, γH2AX, and apoptosis effectors was examined by Western blot. RESULTS: FANCI has copy number variations (CNVs) in OVCA. The high expression of FANCI in OVCA patients was associated with poor survival. Moreover, FANCI expression was correlated with the response to chemotherapy in OVCA. FANCI expression in OVCA cells was induced by carboplatin in a time-dependent manner. Silencing of FANCI had no effect on cell proliferation, but hindered OVCA cell migration and invasion. Mechanically, knockdown of FANCI enhanced DNA damage-induced apoptosis through the CHK1/2-P53-P21 pathway. CONCLUSION: FANCI may be a potential therapeutic target for OVCA patients.
Asunto(s)
Anemia de Fanconi , Neoplasias Ováricas , Carboplatino/farmacología , Carboplatino/uso terapéutico , Carcinoma Epitelial de Ovario , Variaciones en el Número de Copia de ADN , Daño del ADN , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genéticaRESUMEN
The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.
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 , Dimerización , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Mutación , Conformación Proteica , UbiquitinaciónRESUMEN
The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions1. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL2; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown1,3. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.
Asunto(s)
Microscopía por Crioelectrón , ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , ADN/química , Anemia de Fanconi/genética , Humanos , Modelos Moleculares , Conformación Proteica , Ubiquitina/químicaRESUMEN
Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.
Asunto(s)
Avidina/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Antígeno Nuclear de Célula en Proliferación , Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/aislamiento & purificación , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/aislamiento & purificación , Humanos , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Células Sf9 , Spodoptera , Ubiquitina/química , Ubiquitina/aislamiento & purificación , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/aislamiento & purificación , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/aislamiento & purificaciónRESUMEN
Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.
Asunto(s)
Reparación del ADN , ADN/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitina/química , Animales , Sitios de Unión , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Daño del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.
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 , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas/genética , Quinasa de la Caseína II/metabolismo , ADN/metabolismo , Daño del ADN , Reparación del ADN , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Células HeLa , Humanos , Fosforilación , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Guía de Kinetoplastida/metabolismo , Alineación de Secuencia , UbiquitinaciónRESUMEN
Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.
Asunto(s)
Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Histonas/metabolismo , Sitios de Unión , Línea Celular , Cromatina/metabolismo , Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Inestabilidad Genómica , Células HeLa , Histonas/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Growing evidence supports a general hypothesis that aging and cancer are diseases related to energy metabolism. However, the involvement of Fanconi Anemia (FA) signaling, a unique genetic model system for studying human aging or cancer, in energy metabolism remains elusive. Here, we report that FA complementation group D2 protein (FANCD2) functionally impacts mitochondrial ATP production through its interaction with ATP5α, whereas this relationship was not observed in the mutant FANCD2 (K561R)-carrying cells. Moreover, while ATP5α is present within the mitochondria in wild-type cells, it is instead located mostly outside in cells that carry the non-monoubiquitinated FANCD2. In addition, mitochondrial ATP production is significantly reduced in these cells, compared to those cells carrying wtFANCD2. We identified one region (AA42-72) of ATP5α, contributing to the interaction between ATP5α and FANCD2, which was confirmed by protein docking analysis. Further, we demonstrated that mtATP5α (∆AA42-72) showed an aberrant localization, and resulted in a decreased ATP production, similar to what was observed in non-monoubiquitinated FANCD2-carrying cells. Collectively, our study demonstrates a novel role of FANCD2 in governing cellular ATP production, and advances our understanding of how defective FA signaling contributes to aging and cancer at the energy metabolism level.
Asunto(s)
Adenosina Trifosfato/química , Metabolismo Energético/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Mitocondrias/genética , ATPasas de Translocación de Protón Mitocondriales/química , Adenosina Trifosfato/biosíntesis , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Expresión Génica , Células HEK293 , Humanos , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Osteoblastos/citología , Osteoblastos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.
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 , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Espectrometría de Masas , Microscopía Electrónica , Multimerización de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , UbiquitinaciónRESUMEN
During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , N-Glicosil Hidrolasas/metabolismo , Animales , Sistema Libre de Células/química , Reactivos de Enlaces Cruzados/química , ADN/biosíntesis , ADN/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ficusina/química , N-Glicosil Hidrolasas/química , Xenopus laevisRESUMEN
Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , ADN/química , ADN/metabolismo , Reparación del ADN , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de ProteínaRESUMEN
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
Asunto(s)
Proteínas Aviares/metabolismo , Pollos/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Ubiquitinación , Animales , Proteínas Aviares/química , Proteínas Aviares/genética , Línea Celular , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/química , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Humanos , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
DNA interstrand crosslinks (ICLs), highly toxic lesions that covalently link the Watson and Crick strands of the double helix, are repaired by a complex, replication-coupled pathway in higher eukaryotes. The earliest DNA processing event in ICL repair is the incision of parental DNA on either side of the ICL ("unhooking"), which allows lesion bypass. Incisions depend critically on the Fanconi anemia pathway, whose activation involves ubiquitylation of the FANCD2 protein. Using Xenopus egg extracts, which support replication-coupled ICL repair, we show that the 3' flap endonuclease XPF-ERCC1 cooperates with SLX4/FANCP to carry out the unhooking incisions. Efficient recruitment of XPF-ERCC1 and SLX4 to the ICL depends on FANCD2 and its ubiquitylation. These data help define the molecular mechanism by which the Fanconi anemia pathway promotes a key event in replication-coupled ICL repair.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Recombinasas/metabolismo , Animales , Línea Celular , Células Cultivadas , División del ADN , Daño del ADN , Proteínas de Unión al ADN/química , Endodesoxirribonucleasas , Endonucleasas/química , Exodesoxirribonucleasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Humanos , Cinética , Enzimas Multifuncionales , Unión Proteica , Recombinasas/química , Ubiquitinación , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
Asunto(s)
ADN Viral/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitinación , Sustitución de Aminoácidos , Animales , ADN Circular/química , Proteína del Grupo de Complementación L de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/química , Nucleosomas/química , Plásmidos/química , Unión Proteica , Células Sf9 , Spodoptera , Especificidad por SustratoRESUMEN
Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
Asunto(s)
Núcleo Celular/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Señales de Localización Nuclear , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatina/metabolismo , Secuencia Conservada , Daño del ADN , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Transporte de Proteínas , Alineación de Secuencia , UbiquitinaciónRESUMEN
How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Línea Celular , Cromatina/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Células HeLa , Histonas/metabolismo , Humanos , Mitomicina/farmacología , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Mutación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/efectos de la radiación , Estructura Terciaria de Proteína , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/efectos de la radiación , Rayos UltravioletaRESUMEN
The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.
Asunto(s)
Proteína del Grupo de Complementación L de la Anemia de Fanconi/química , Anemia de Fanconi/enzimología , Pliegue de Proteína , Animales , Drosophila melanogaster , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , 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 L de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Humanos , Mutación , Mapeo Peptídico , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the ~300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , Ubiquitina/química , UbiquitinaciónRESUMEN
A conserved DNA repair response is defective in the human genetic illness Fanconi anemia (FA). Mutation of some FA genes impairs homologous recombination and error-prone DNA repair, rendering FA cells sensitive to DNA cross-linking agents. We found a genetic interaction between the FA gene FANCC and the nonhomologous end joining (NHEJ) factor Ku70. Disruption of both FANCC and Ku70 suppresses sensitivity to cross-linking agents, diminishes chromosome breaks, and reverses defective homologous recombination. Ku70 binds directly to free DNA ends, committing them to NHEJ repair. We show that purified FANCD2, a downstream effector of the FA pathway, might antagonize Ku70 activity by modifying such DNA substrates. These results reveal a function for the FA pathway in processing DNA ends, thereby diverting double-strand break repair away from abortive NHEJ and toward homologous recombination.