RESUMEN
Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.
Asunto(s)
ADN Cruciforme , Transferencia Resonante de Energía de Fluorescencia , ADN Cruciforme/genética , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/química , Factores de Integración del Huésped/metabolismo , Conformación de Ácido Nucleico , ADN ViralRESUMEN
Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/análogos & derivados , Azacitidina/farmacología , Azacitidina/uso terapéutico , Médula Ósea/patología , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Decitabina/farmacología , Decitabina/uso terapéutico , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Selección de Paciente , Cultivo Primario de Células , Estudios Retrospectivos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5' external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5' external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5' external transcribed spacer ribonucleoprotein - provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.
Asunto(s)
Precursores del ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , Células Eucariotas/metabolismo , Modelos Moleculares , Conformación Molecular , Dominios Proteicos , Precursores del ARN/química , Precursores del ARN/genética , ARN Ribosómico 18S/química , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas/química , Subunidades Ribosómicas Pequeñas/metabolismo , Subunidades Ribosómicas Pequeñas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.
Asunto(s)
Metilación de ADN/fisiología , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 4/fisiología , Regiones Promotoras Genéticas/fisiología , Transactivadores/metabolismo , Activación Viral/fisiología , Secuencias de Aminoácidos , Línea Celular Tumoral , ADN Viral/genética , Células HEK293 , Humanos , Mutación , Dominios Proteicos , Transactivadores/genéticaRESUMEN
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Interacciones Farmacológicas/fisiología , Leucemia Mieloide Aguda/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Nucleótidos/farmacología , Especificidad por SustratoRESUMEN
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells.IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Transcripción Genética/fisiología , Latencia del Virus/fisiología , Sustitución de Aminoácidos , Linfocitos T CD4-Positivos/virología , Células HEK293 , Humanos , Células Jurkat , Mutación Missense , Proteína 1 que Contiene Dominios SAM y HD/genética , Células THP-1RESUMEN
Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.
Asunto(s)
Inmunidad Innata , Inflamación/inmunología , Interferón-alfa/biosíntesis , FN-kappa B/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/fisiología , Virosis/inmunología , Animales , Células Cultivadas , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Células HEK293 , VIH/fisiología , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Factor 7 Regulador del Interferón/antagonistas & inhibidores , Interferón-alfa/genética , Macrófagos/inmunología , Macrófagos/virología , Masculino , Ratones , Inhibidor NF-kappaB alfa/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/inmunología , Virus Sendai/fisiología , Transducción de Señal/inmunología , Células THP-1RESUMEN
Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis-for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.
Asunto(s)
Desaminasa APOBEC-3G/química , Desaminasa APOBEC-3G/metabolismo , ADN de Cadena Simple/metabolismo , Desaminasa APOBEC-3G/genética , Sitios de Unión , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
Human SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular nucleotides required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory SAM domain. Here we report the crystal structure of full-length SAMHD1 by capturing mouse SAMHD1 (mSAMHD1) structures in three different nucleotide bound states. Although mSAMHD1 and hSAMHD1 are highly similar in sequence and function, we find that mSAMHD1 possesses a more complex nucleotide-induced activation process, highlighting the regulatory role of the SAM domain. Our results provide insights into the regulation of SAMHD1 activity, thereby facilitating the improvement of HIV mouse models and the development of new therapies for certain cancers and autoimmune diseases.
Asunto(s)
Proteína 1 que Contiene Dominios SAM y HD/química , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Sitio Alostérico , Animales , Cristalografía por Rayos X , Hidrólisis , Ratones , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.
Asunto(s)
Infecciones por VIH , VIH-1 , Mutación Missense , Multimerización de Proteína , Proteína 1 que Contiene Dominios SAM y HD , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Células HEK293 , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , VIH-1/química , VIH-1/genética , VIH-1/metabolismo , Humanos , Fosforilación , Dominios Proteicos , Proteína 1 que Contiene Dominios SAM y HD/química , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Relación Estructura-Actividad , Células THP-1 , Células U937RESUMEN
The S. cerevisiae Pif1 helicase functions with DNA polymerase (Pol) δ in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction. This mutation impairs the ability of Pif1 to enhance DNA strand displacement synthesis by Pol δ in vitro and also the efficiency of BIR in cells. These results provide insights into the role of the Pif1-PCNA-Pol δ ensemble during DNA break repair by homologous recombination.
Asunto(s)
ADN Helicasas/química , ADN Polimerasa III/química , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/química , Proteínas de Saccharomyces cerevisiae/química , Sitios de Unión , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , ADN Polimerasa III/metabolismo , Reparación del ADN , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance.
Asunto(s)
Emparejamiento Cromosómico , ADN/metabolismo , Recombinación Homóloga , Recombinasa Rad51/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
PURPOSE: Retinal dystrophies (RD) are heterogeneous hereditary disorders of the retina that are usually progressive in nature. The aim of this study was to clinically and molecularly characterize a large cohort of RD patients. METHODS: We have developed a next-generation sequencing assay that allows known RD genes to be sequenced simultaneously. We also performed mapping studies and exome sequencing on familial and on syndromic RD patients who tested negative on the panel. RESULTS: Our panel identified the likely causal mutation in >60% of the 292 RD families tested. Mapping studies on all 162 familial RD patients who tested negative on the panel identified two novel disease loci on Chr2:25,550,180-28,794,007 and Chr16:59,225,000-72,511,000. Whole-exome sequencing revealed the likely candidate as AGBL5 and CDH16, respectively. We also performed exome sequencing on negative syndromic RD cases and identified a novel homozygous truncating mutation in GNS in a family with the novel combination of mucopolysaccharidosis and RD. Moreover, we identified a homozygous truncating mutation in DNAJC17 in a family with an apparently novel syndrome of retinitis pigmentosa and hypogammaglobulinemia. CONCLUSION: Our study expands the clinical and allelic spectrum of known RD genes, and reveals AGBL5, CDH16, and DNAJC17 as novel disease candidates.Genet Med 18 6, 554-562.
Asunto(s)
Cadherinas/genética , Carboxipeptidasas/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Distrofias Retinianas/genética , Femenino , Homocigoto , Humanos , Masculino , Mutación , Linaje , Fenotipo , Retina/patología , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/patología , Secuenciación del ExomaRESUMEN
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.