RESUMEN
Kaposi-sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is the causative agent of several malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Active KSHV replication has also been associated with a pathological condition called KSHV inflammatory cytokine syndrome (KICS), and KSHV may play a role in rare cases of post-transplant polyclonal lymphoproliferative disorders. Several commonly used herpesviral DNA polymerase inhibitors are active against KSHV in tissue culture. Unfortunately, they are not always efficacious against KSHV-induced diseases. To improve the outcome for the patients, new therapeutics need to be developed, including treatment strategies that target either viral proteins or cellular pathways involved in tumor growth and/or supporting the viral life cycle. In this review, we summarize the most commonly established treatments against KSHV-related diseases and review recent developments and promising new compounds that are currently under investigation or on the way to clinical use.
Asunto(s)
Infecciones por Herpesviridae/tratamiento farmacológico , Herpesvirus Humano 8/efectos de los fármacos , Sarcoma de Kaposi/tratamiento farmacológico , Replicación Viral/genética , Animales , Sistemas CRISPR-Cas , Enfermedad de Castleman/tratamiento farmacológico , Ensayos Clínicos como Asunto , ADN Polimerasa Dirigida por ADN , Exodesoxirribonucleasas/antagonistas & inhibidores , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/clasificación , Herpesvirus Humano 8/genética , Humanos , Linfoma de Efusión Primaria/tratamiento farmacológico , Ratones , Sarcoma de Kaposi/virología , Proteínas Virales/antagonistas & inhibidores , Latencia del Virus/genética , Replicación Viral/efectos de los fármacosRESUMEN
PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like toll-like receptor 9, and PLD4-deficent mice exhibit inflammatory disease. Targeting these immunoregulatory enzymes presents an opportunity to indirectly regulate innate immune nucleic acid sensors that could yield immunotherapies, adjuvants, and nucleic acid drug stabilizers. To aid in delineating the therapeutic potential of these targets, we have developed a high-throughput fluorescence enzymatic assay to identify modulators of PLD3 and PLD4. Screening of a diversity library (N = 17952) yielded preferential inhibitors of PLD3 and PLD4 in addition to a PLD3 selective activator. The modulation models of these compounds were delineated by kinetic analysis. This work presents an inexpensive and simple method to identify modulators of these immunoregulatory exonucleases.
Asunto(s)
Activadores de Enzimas/química , Inhibidores Enzimáticos/química , Exodesoxirribonucleasas/antagonistas & inhibidores , Fosfolipasa D/antagonistas & inhibidores , Pruebas de Enzimas , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Nitrofenoles/química , Nucleótidos de Timina/química , Umbeliferonas/químicaRESUMEN
SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5'-position or 3'-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5'-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Nucleósidos/farmacología , Compuestos Organometálicos/farmacología , Sitios de Unión/efectos de los fármacos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ésteres/química , Ésteres/farmacología , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Malonatos/química , Malonatos/farmacología , Estructura Molecular , Nucleósidos/química , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/químicaRESUMEN
Antimicrobial resistance is a pressing threat to global health, with multidrug-resistant pathogens becoming increasingly prevalent. The bacterial SOS pathway functions in response to DNA damage that occurs during infection, initiating several pro-survival and resistance mechanisms, such as DNA repair and hypermutation. This makes SOS pathway components potential targets that may combat drug-resistant pathogens and decrease resistance emergence. This review discusses the mechanism of the SOS pathway; the structure and function of potential targets AddAB, RecBCD, RecA and LexA; and efforts to develop selective small-molecule inhibitors of these proteins. These inhibitors may serve as valuable tools for target validation and provide the foundations for desperately needed novel antibacterial therapeutics.
Asunto(s)
Antibacterianos/química , Inhibidores Enzimáticos/química , Respuesta SOS en Genética/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Reparación del ADN/efectos de los fármacos , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasa V/antagonistas & inhibidores , Exodesoxirribonucleasa V/genética , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Regulación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Rec A Recombinasas/antagonistas & inhibidores , Rec A Recombinasas/genética , Serina Endopeptidasas/genética , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Ácidos Hidroxámicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Exodesoxirribonucleasas/metabolismo , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Genotypic diagnosis of HSV drug resistance can be performed routinely in a clinically relevant time. Nevertheless, data about HSV mutations (polymorphism or resistance) is not exhaustive which hinders the interpretation of such tests. The UL23, UL30, and UL5 genes are of greatest interest as these encode, respectively, thymidine kinase, DNA polymerase, and helicase, which, if mutated may affect the effectiveness of acyclovir, foscarnet, cidofovir, and helicase-primase inhibitors. The present study aimed to extensively characterize UL23, UL30, and UL5 genes. A total of 239 clinical HSV1 recovered from patients admitted to the hematology departments of the Lyon teaching hospitals were included in this single-center retrospective study. Drug resistance was evaluated using the neutral red dye-uptake assay, and sequencing using the Sanger method. Additional information on HSV1 natural polymorphism and resistance is now available. Twenty-two amino acid substitutions related to polymorphism were described on UL23 (E43A, L50M, L68R, Q109K, A133V, A136N, S150L, D258N, S263L, P280S, N301S, A316S, M322L, I326V, D330A, D338H, Q342H, T344I, Q349R, V352L, R370W, E371D), and 6â¯amino acid substitutions on UL30 (G641R, G645D, E649G, G679D, R681L, I966M). Moreover, the UL23 substitution L242P was added to ACV resistance-related mutations. There were 12 substitutions on UL23 (A37S, V70M, S74L, H151N, P154S, P155Q, L159R, E225L, Y248H, Q270R, N303Y, M372I), and 8 on UL5 (L49I, L138V, S173L, A280T, A575V, V600A, A602T, D862N) that remain of unclear significance with regards to drug resistance. The mean (±standard deviation, SD) number of natural polymorphisms in UL23 was 2.53 (±2.55), in UL30 it was 0.83 (±1.02), and in UL5 it was 5.00 (±1.59) There was no association between HSV1 phenotype and the frequency of substitutions. The results reported herein provide valuable new information concerning HSV1 mutations that will assist the interpretation of genotypic assays.
Asunto(s)
Herpes Simple/microbiología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/aislamiento & purificación , Proteínas Virales/genética , Antivirales/farmacología , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN Primasa/antagonistas & inhibidores , ADN Primasa/genética , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Femenino , Genotipo , Hematología , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/clasificación , Humanos , Masculino , Mutación , Filogenia , Polimorfismo Genético , Estudios Retrospectivos , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/genética , Proteínas Virales/antagonistas & inhibidoresRESUMEN
Acyclic nucleoside phosphonates represent a well-defined class of clinically used nucleoside analogs. All acyclic nucleoside phosphonates need intracellular phosphorylation before they can bind viral DNA polymerases. Recently, a novel class of alpha-carboxynucleoside phosphonates have been designed to mimic the natural 2'-deoxynucleotide 5'-triphosphate substrates of DNA polymerases. They contain a carboxyl group in the phosphonate moiety linked to the nucleobase through a cyclic or acyclic bridge. Alpha-carboxynucleoside phosphonates act as viral DNA polymerase inhibitors without any prior requirement of metabolic conversion. Selective inhibitory activity against retroviral reverse transcriptase and herpesvirus DNA polymerases have been demonstrated. These compounds have a unique mechanism of inhibition of viral DNA polymerases, and provide possibilities for further modifications to optimize and fine tune their antiviral DNA polymerase spectrum.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Nucleósidos/análogos & derivados , Nucleósidos/farmacología , Organofosfonatos/química , Organofosfonatos/farmacología , Animales , ADN Polimerasa Dirigida por ADN , Descubrimiento de Drogas , Exodesoxirribonucleasas/antagonistas & inhibidores , Herpes Simple/tratamiento farmacológico , Humanos , Modelos Moleculares , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Proteínas Virales/antagonistas & inhibidores , Virosis/tratamiento farmacológico , Virus/efectos de los fármacos , Virus/enzimologíaRESUMEN
Viral accessory proteins hijack host cell E3 ubiquitin ligases to antagonize innate/intrinsic defenses and thereby provide a more permissive environment for virus replication. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes, but the significance and role of these Vpr interactions are poorly understood. To gain additional insights, we performed a focused screen for substrates of CRL4DCAF1 E3 reprogrammed by HIV-1 Vpr among known postreplication DNA repair proteins and identified exonuclease 1 (Exo1) as a novel direct HIV-1 Vpr target. We show that HIV-1 Vpr recruits Exo1 to the CRL4DCAF1 E3 complex for ubiquitination and subsequent proteasome-dependent degradation and that Exo1 levels are depleted in HIV-1-infected cells in a Vpr-dependent manner. We also show that Exo1 inhibits HIV-1 replication in T cells. Notably, the antagonism of Exo1 is a conserved function of main group HIV-1 and its ancestor Vpr proteins in the simian immunodeficiency virus from chimpanzee (SIVcpz) lineage, further underscoring the relevance of our findings. Overall, our studies (i) reveal that HIV-1 Vpr extensively remodels the cellular postreplication DNA repair machinery by impinging on multiple repair pathways, (ii) support a model in which Vpr promotes HIV-1 replication by antagonizing select DNA repair enzymes, and (iii) highlight the importance of a new class of restrictions placed on HIV-1 replication in T cells by the cellular DNA repair machinery.IMPORTANCE HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA "repair" inhibit HIV-1 infection.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Interacciones Huésped-Patógeno , Ubiquitina-Proteína Ligasas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Infecciones por VIH/enzimología , Infecciones por VIH/genética , VIH-1/genética , VIH-1/fisiología , Humanos , Linfocitos T/virología , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy numbers were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quantitative PCR and QuantiGene® 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy number variation (CNV) of the Exo1 gene was found in HCC specimens in three separate sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addition, EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradiation. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.
Asunto(s)
Carcinoma Hepatocelular/patología , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de la radiación , Variaciones en el Número de Copia de ADN , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Radiación IonizanteRESUMEN
Background: Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. Methods: HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Results: Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Conclusions: Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy.
Asunto(s)
Antivirales/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Tenofovir/farmacología , Proteínas Virales/antagonistas & inhibidores , Aciclovir/farmacología , Sustitución de Aminoácidos , Células Cultivadas , Análisis Mutacional de ADN , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Viral , Exodesoxirribonucleasas/genética , Foscarnet/farmacología , Herpes Genital/virología , Herpes Simple/virología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/aislamiento & purificación , Humanos , Mutación Missense , Organofosfonatos/farmacología , Pirimidinas/farmacología , Selección Genética , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
Maintaining genome integrity is important for cells and damaged DNA triggers autoimmunity. Previous studies have reported that Three-prime repair exonuclease 1(TREX1), an endogenous DNA exonuclease, prevents immune activation by depleting damaged DNA, thus preventing the development of certain autoimmune diseases. Consistently, mutations in TREX1 are linked with autoimmune diseases such as systemic lupus erythematosus, Aicardi-Goutières syndrome (AGS) and familial chilblain lupus. However, TREX1 mutants competent for DNA exonuclease activity are also linked to AGS. Here, we report a nuclease-independent involvement of TREX1 in preventing the L1 retrotransposon-induced DNA damage response. TREX1 interacted with ORF1p and altered its intracellular localization. Furthermore, TREX1 triggered ORF1p depletion and reduced the L1-mediated nicking of genomic DNA. TREX1 mutants related to AGS were deficient in inducing ORF1p depletion and could not prevent L1-mediated DNA damage. Therefore, our findings not only reveal a new mechanism for TREX1-mediated L1 suppression and uncover a new function for TREX1 in protein destabilization, but they also suggest a novel mechanism for TREX1-mediated suppression of innate immune activation through maintaining genome integrity.
Asunto(s)
ADN/genética , Exodesoxirribonucleasas/genética , Genoma Humano , Fosfoproteínas/genética , Proteínas/genética , Retroelementos , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/patología , Autoinmunidad , ADN/inmunología , Roturas del ADN de Doble Cadena , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/inmunología , Regulación de la Expresión Génica , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/patología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/inmunología , Fosforilación , Plásmidos/química , Plásmidos/metabolismo , Proteínas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , TransfecciónRESUMEN
Bacterial metallo-ß-lactamases (MBLs) are involved in resistance to ß-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact ß-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.
Asunto(s)
Cefalosporinas/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , beta-Lactamasas/metabolismo , Proteínas de Ciclo Celular , Cefalosporinas/síntesis química , Cefalosporinas/química , Enzimas Reparadoras del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Exodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Conformación Molecular , Proteínas Nucleares/metabolismo , Relación Estructura-ActividadRESUMEN
DNA double-strand break (DSB) repair by homologous recombination (HR) involves resection of the break to expose a 3' single-stranded DNA tail. In budding yeast, resection occurs in two steps: initial short-range resection, performed by Mre11-Rad50-Xrs2 and Sae2; and long-range resection catalysed by either Exo1 or Sgs1-Dna2. Here we use genetic assays to investigate the importance of Exo1 and the Sgs1 homologue Rqh1 for DNA repair and promotion of direct repeat recombination in the fission yeast Schizosaccharomyces pombe. We find that Exo1 and Rqh1 function in alternative redundant pathways for promoting survival following replication fork breakage. Exo1 promotes replication fork barrier-induced direct repeat recombination but intriguingly limits recombination induced by fork breakage. Direct repeat recombination induced by ultraviolet light depends on either Exo1 or Rqh1. Finally, we show that Rqh1 plays a major role in limiting Exo1-dependent direct repeat recombination induced by replication fork stalling but only a minor role in constraining recombination induced by fork breakage. The implications of our findings are discussed in the context of the benefits that long-range resection may bring to processing perturbed replication forks.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Exodesoxirribonucleasas/antagonistas & inhibidores , Recombinación Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Reparación del ADNRESUMEN
We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.
Asunto(s)
Aciclovir/farmacología , Antivirales/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Foscarnet/farmacología , Herpesvirus Humano 1/enzimología , Modelos Moleculares , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas Virales/antagonistas & inhibidores , Aciclovir/química , Aciclovir/metabolismo , Antivirales/química , Antivirales/metabolismo , Dominio Catalítico , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Foscarnet/química , Foscarnet/metabolismo , Herpesvirus Humano 1/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Cinética , Estructura Molecular , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Especificidad por Sustrato , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
A novel enzyme inhibition-based homogeneous electrochemical biosensing strategy was designed for an organophosphorus pesticide assay based on exploiting the resistance of a mercury ion-mediated helper probe (HP) toward nuclease-catalyzed digestion and the remarkable diffusivity difference between HPs and the mononucleotides toward a negatively charged indium tin oxide (ITO) electrode. In particular, the mercury ion-mediated T-Hg(2+)-T base pairs facilitate the HP labeled with methylene blue (MB) to fold into a hairpin structure, preventing its digestion by exonuclease I, and thus resulting in a low electrochemical response because of the large electrostatic repulsion between the negatively charged ITO electrode and the HPs. The competitive binding by a thiol group (-SH), produced in the hydrolysis reaction of acetylthiocholine (ACh) chloride with acetylcholinesterase (AChE), removes mercury ions from the base pairs, causing a nuclease-catalyzed digestion, and the subsequent electrochemical response increase due to the weak electrostatic repulsion between the product-mononucleotides and the ITO electrode. Mercury ion-mediated HPs were first designed for pesticide detection and diazinon was chosen as the model target. Under the optimal experimental conditions, the approach exhibited high sensitivity for diazinon detection with a detection limit of 0.25 µg L(-1). The satisfactory results in the determination of diazinon in real samples demonstrate that the method possesses great potential for detecting organophosphorus pesticides. This new approach is expected to promote the exploitation of mercury-mediated base pair-based homogenous electrochemical biosensors in biochemical studies and in the food safety field.
Asunto(s)
Biocatálisis , Técnicas Biosensibles/métodos , ADN/química , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/metabolismo , Conformación de Ácido Nucleico , Compuestos Organofosforados/análisis , Animales , Técnicas Biosensibles/instrumentación , Electroquímica , Electrophorus , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Diseño de Equipo , Estudios de Factibilidad , Inocuidad de los Alimentos , Límite de Detección , Compuestos Organofosforados/farmacología , Residuos de Plaguicidas/análisis , Residuos de Plaguicidas/farmacología , Plaguicidas/análisis , Plaguicidas/farmacología , Agua/químicaRESUMEN
Human WRN, a RecQ helicase encoded by the Werner syndrome gene, is implicated in genome maintenance, including replication, recombination, excision repair and DNA damage response. These genetic processes and expression of WRN are concomitantly upregulated in many types of cancers. Therefore, targeted destruction of this helicase could be useful for elimination of cancer cells. Here, we provide a proof of concept for applying the external guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive 3'-5' DNA helicase-nuclease. Remarkably, EGS-directed knockdown of WRN leads to severe inhibition of cell viability. Hence, further assessment of this targeting system could be beneficial for selective cancer therapies, particularly in the light of the recent improvements introduced into EGSs.
Asunto(s)
Exodesoxirribonucleasas/genética , Biosíntesis de Proteínas , RecQ Helicasas/genética , Ribonucleasa P/genética , Síndrome de Werner/genética , Línea Celular , Daño del ADN , Reparación del ADN/genética , Replicación del ADN/genética , Exodesoxirribonucleasas/antagonistas & inhibidores , Genoma Humano , Inestabilidad Genómica/genética , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , RecQ Helicasas/antagonistas & inhibidores , Síndrome de Werner/patología , Helicasa del Síndrome de WernerRESUMEN
TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.
Asunto(s)
Citosol/enzimología , Exodesoxirribonucleasas/metabolismo , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Aclarubicina/análogos & derivados , Aclarubicina/farmacología , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Mutación del Sistema de Lectura , Células HEK293 , Células HeLa , Hexosiltransferasas/genética , Humanos , Inmunidad Innata/genética , Immunoblotting , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Polisacáridos/metabolismo , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3'-5' exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/fisiología , Línea Celular Tumoral , Aberraciones Cromosómicas , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Exodesoxirribonucleasas/antagonistas & inhibidores , Humanos , Proteína Homóloga de MRE11 , Mutación , Pirimidinonas/farmacología , Eliminación de Secuencia , Telómero , Tionas/farmacologíaRESUMEN
4-Hydroxy-2-nonenal (HNE) is a reactive α,ß-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.
Asunto(s)
Aldehídos/química , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , RecQ Helicasas/química , RecQ Helicasas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Aldehídos/análisis , Animales , Biocatálisis , Células Cultivadas , ADN/metabolismo , Exodesoxirribonucleasas/antagonistas & inhibidores , Humanos , Modelos Moleculares , RecQ Helicasas/antagonistas & inhibidores , Helicasa del Síndrome de WernerRESUMEN
TREX-2 is a five protein complex, conserved from yeast to humans, involved in linking mRNA transcription and export. The centrin 2 subunit of TREX-2 is also a component of the centrosome and is additionally involved in a distinctly different process of nuclear protein export. While centrin 2 is a known multifunctional protein, the roles of other human TREX-2 complex proteins other than mRNA export are not known. In this study, we found that human TREX-2 member PCID2 but not ENY2 is involved in some of the same cellular processes as those of centrin 2 apart from the classical TREX-2 function. PCID2 is present at the centrosome in a subset of HeLa cells and this localization is centrin 2 dependent. Furthermore, the presence of PCID2 at the centrosome is prevalent throughout the cell cycle as determined by co-staining with cyclins E, A and B. PCID2 but not ENY2 is also involved in protein export. Surprisingly, siRNA knockdown of PCID2 delayed the rate of nuclear protein export, a mechanism distinct from the effects of centrin 2, which when knocked down inhibits export. Finally we showed that co-depletion of centrin 2 and PCID2 leads to blocking rather than delaying nuclear protein export, indicating the dominance of the centrin 2 phenotype. Together these results represent the first discovery of specific novel functions for PCID2 other than mRNA export and suggest that components of the TREX-2 complex serve alternative shared roles in the regulation of nuclear transport and cell cycle progression.