RESUMEN
R-loops are three-stranded RNA-DNA hybrid structures that play important regulatory roles, but excessive or deregulated R-loops formation can trigger DNA damage and genome instability. Digestion of R-loops is mainly relying on the action of two specialized ribonucleases: RNaseH1 and RNaseH2. RNaseH2 is the main enzyme carrying out the removal of misincorporated rNMPs during DNA replication or repair, through the Ribonucleotide Excision Repair (RER) pathway. We have recently shown that the human RNA helicase DDX3X possessed RNaseH2-like activity, being able to substitute RNaseH2 in reconstituted RER reactions. Here, using synthetic R-loop mimicking substrates, we could show that human DDX3X alone was able to both displace and degrade the ssRNA strand hybridized to DNA. Moreover, DDX3X was found to physically interact with human RNaseH2. Such interaction suppressed the nuclease and helicase activities of DDX3X, but stimulated severalfold the catalytic activity of the trimeric RNaseH2, but not of RNaseH1. Finally, silencing of DDX3X in human cells caused accumulation of RNA-DNA hybrids and phosphorylated RPA foci. These results support a role of DDX3X as a scaffolding protein and auxiliary factor for RNaseH2 during R-loop degradation.
RESUMEN
Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.
Asunto(s)
Ataxia Cerebelosa , Enfermedades del Sistema Nervioso Periférico , Síndrome , Enfermedades Vestibulares , Humanos , Vestibulopatía Bilateral , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/diagnóstico , Enfermedades Neurodegenerativas , Enfermedades del Sistema Nervioso Periférico/diagnóstico , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades Vestibulares/diagnóstico , Enfermedades Vestibulares/genéticaRESUMEN
The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.
Asunto(s)
Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/químicaRESUMEN
Inhibition of c-Src is considered one of the most studied approaches to cancer treatment, with several heterocyclic compounds approved during the last 15 years as chemotherapeutic agents. Starting from the biological evaluation of an in-house collection of small molecules, indolinone was selected as the most promising scaffold. In this work, several functionalised indolinones were synthesised and their inhibitory potency and cytotoxic activity were assayed. The pharmacological profile of the most active compounds, supported by molecular modelling studies, revealed that the presence of an amino group increased the affinity towards the ATP-binding site of c-Src. At the same time, bulkier derivatizations seemed to improve the interactions within the enzymatic pocket. Overall, these data represent an early stage towards the optimisation of new, easy-to-be functionalised indolinones as potential c-Src inhibitors.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Antineoplásicos/química , Simulación del Acoplamiento Molecular , Oxindoles , Proteínas Tirosina Quinasas , Relación Estructura-ActividadRESUMEN
Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.
Asunto(s)
Replicación del ADN , ADN/química , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Ribonucleótidos/química , Catálisis , HumanosRESUMEN
Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
Asunto(s)
Aminopeptidasas/metabolismo , ADN Polimerasa beta/metabolismo , G-Cuádruplex , Serina Proteasas/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismo , Línea Celular Tumoral , Humanos , Complejos Multiproteicos , Proteína de Replicación A/metabolismo , Complejo Shelterina , Telómero/química , Telómero/metabolismoRESUMEN
G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.
Asunto(s)
Complejos de Coordinación/química , Cobre/química , G-Cuádruplex , Imidas/química , Naftalenos/química , Sitios de Unión , DEET/química , Ligandos , Oxidación-Reducción , Polietilenglicoles/química , Relación Estructura-ActividadRESUMEN
p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.
Asunto(s)
Proteínas Portadoras/química , ADN/química , Proteínas Intrínsecamente Desordenadas/química , Antígeno Nuclear de Célula en Proliferación/química , Proteínas Portadoras/genética , Cristalografía por Rayos X , ADN/genética , ADN Polimerasa III/química , ADN Polimerasa III/genética , Replicación del ADN/genética , Proteínas de Unión al ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Espectroscopía de Resonancia Magnética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Antígeno Nuclear de Célula en Proliferación/genéticaRESUMEN
Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2Î-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Ribonucleasa H/metabolismo , Ribonucleótidos/metabolismo , Línea Celular , Citidina Monofosfato/metabolismo , ADN/biosíntesis , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , ARN/biosíntesis , Xerodermia Pigmentosa/genéticaRESUMEN
The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proto-Oncogenes Mas , Pirazoles/síntesis química , Pirazoles/química , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Infections by the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), are still totaling an appalling 36.7 millions worldwide, with 1.1 million AIDS deaths/year and a similar number of yearly new infections. All this, in spite of the discovery of HIV-1 as the AIDS etiological agent more than 30 years ago and the introduction of an effective combinatorial antiretroviral therapy (cART), able to control disease progression, more than 20 years ago. Although very effective, current cART is plagued by the emergence of drug-resistant viral variants and most of the efforts in the development of novel direct-acting antiviral agents (DAAs) against HIV-1 have been devoted toward the fighting of resistance. In this review, rather than providing a detailed listing of all the drugs and the corresponding resistance mutations, we aim, through relevant examples, at presenting to the general reader the conceptual shift in the approaches that are being taken to overcome the viral resistance hurdle. From the classic 'running faster' strategy, based on the development of novel DAAs active against the mutant viruses selected by the previous drugs and/or presenting to the virus a high genetic barrier toward the development of resilience, to a 'jumping higher' approach, which looks at the cell, rather than the virus, as a source of valuable drug targets, in order to make the cellular environment non-permissive toward the replication of both wild-type and mutated viruses.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Diseño de Fármacos , Farmacorresistencia Viral Múltiple , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Modelos Biológicos , Animales , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa/efectos adversos , Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/farmacología , Antagonistas de los Receptores CCR5/uso terapéutico , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Quimioterapia Combinada/efectos adversos , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/efectos adversos , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Estructura Molecular , Terapia Molecular Dirigida , Mutación , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Fenómenos Fisiológicos de los Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.
Asunto(s)
ADN/metabolismo , Conformación de Ácido Nucleico , Ribonucleótidos/metabolismo , ADN/química , Escherichia coli , Modelos Lineales , Microscopía de Fuerza Atómica , Mutación , Reacción en Cadena de la Polimerasa , Ribonucleótidos/química , Polimerasa Taq/genética , Polimerasa Taq/metabolismoRESUMEN
The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Miristatos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Membrana/genética , Modelos Moleculares , Estructura Molecular , Mutación , Miristatos/síntesis química , Miristatos/química , Proteínas de Neoplasias/genética , Relación Estructura-ActividadRESUMEN
The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors.
Asunto(s)
Mutación Puntual , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Adenosina Trifosfato/metabolismo , Diseño de Fármacos , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/enzimología , Tumores del Estroma Gastrointestinal/genética , Humanos , Cinética , Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol δ-interacting protein 2 (PolDIP2) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol δ (family B), Pol η and Pol ι (family Y), and Pol λ and Pol ß (family X). Our results show that PolDIP2 also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols ß or ι. Additionally, we provide evidence that PolDIP2 stimulates Pol δ without affecting its fidelity, facilitating the switch from Pol δ to Pol λ during 8-oxo-G TLS. PolDIP2 stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP2 is an important mediator for TLS.
Asunto(s)
Daño del ADN/genética , ADN Polimerasa beta/metabolismo , Replicación del ADN/fisiología , Guanina/análogos & derivados , Proteínas Nucleares/metabolismo , Cromatografía por Intercambio Iónico , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Fluorescencia , Guanina/metabolismo , Humanos , Inmunoprecipitación , Cinética , Oligonucleótidos/genética , ARN Interferente Pequeño/genéticaRESUMEN
The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Daño del ADN , ADN Polimerasa beta/metabolismo , Estrés Oxidativo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Arabidopsis/metabolismo , Clonación Molecular , ADN de Plantas/metabolismo , Guanina/análogos & derivados , Guanina/química , Humanos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Protoplastos/metabolismoRESUMEN
'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols ß, λ and µ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol ß, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol ß. We propose a possible novel role of DNA pol ß in MMEJ, promoting (CAG)n triplet repeats instability.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Polimerasa beta/metabolismo , ADN/biosíntesis , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , ADN/química , ADN/metabolismo , ADN Polimerasa beta/química , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Proteína Huntingtina , Proteínas del Tejido Nervioso/genética , Fosfatos/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteína de Replicación A/metabolismo , Homología de Secuencia de Ácido Nucleico , Moldes Genéticos , Expansión de Repetición de Trinucleótido , Repeticiones de TrinucleótidosRESUMEN
Respiratory tract infections involving a variety of microorganisms such as viruses, bacteria, and fungi are a prominent cause of morbidity and mortality globally, exacerbating various pre-existing respiratory and non-respiratory conditions. Moreover, the ability of bacteria and viruses to coexist might impact the development and severity of lung infections, promoting bacterial colonization and subsequent disease exacerbation. Secondary bacterial infections following viral infections represent a complex challenge to be overcome from a therapeutic point of view. We report herein our efforts in the development of new bithiazole derivatives showing broad-spectrum antimicrobial activity against both viruses and bacteria. A series of 4-trifluoromethyl bithiazole analogues was synthesized and screened against selected viruses (hRVA16, EVD68, and ZIKV) and a panel of Gram-positive and Gram-negative bacteria. Among them, two promising broad-spectrum antimicrobial compounds (8a and 8j) have been identified: both compounds showed low micromolar activity against all tested viruses, 8a showed synergistic activity against E. coli and A. baumannii in the presence of a subinhibitory concentration of colistin, while 8j showed a broader spectrum of activity against Gram-positive and Gram-negative bacteria. Activity against antibiotic-resistant clinical isolates is also reported. Given the ever-increasing need to adequately address viral and bacterial infections or co-infections, this study paves the way for the development of new agents with broad antimicrobial properties and synergistic activity with common antivirals and antibacterials.
RESUMEN
Replicative DNA polymerases (DNA pols) increase their fidelity by removing misincorporated nucleotides with their 3' â 5' exonuclease activity. Exonuclease activity reduces translesion synthesis (TLS) efficiency and TLS DNA pols lack 3' â 5' exonuclease activity. Here we show that physiological concentrations of pyrophosphate (PP(i)) activate the pyrophosphorolytic activity by DNA pol-λ, allowing the preferential excision of the incorrectly incorporated A opposite a 7,8-dihydro-8-oxoguanine lesion, or T opposite a 6-methyl-guanine, with respect to the correct C. This is the first example of an alternative proofreading mechanism used during TLS.
Asunto(s)
ADN Polimerasa beta/metabolismo , Roturas del ADN de Cadena Simple , Reparación del ADN , Replicación del ADN/fisiología , Nucleótidos de Desoxiadenina/metabolismo , Difosfatos/metabolismo , Activación Enzimática , Guanosina/análogos & derivados , Guanosina/metabolismo , HumanosRESUMEN
Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.