RESUMO
A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low- and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.
Assuntos
COVID-19 , Nanopartículas de Magnetita , Animais , Humanos , RNA Viral/análise , SARS-CoV-2/genética , DNA , Sensibilidade e Especificidade , Mamíferos/genéticaRESUMO
The year of 2020 was profoundly marked by a global pandemic caused by a strain of coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19). To control disease spread, a key strategy adopted by many countries was the regular testing of individuals for infection. This led to the rapid development of diagnostic testing technologies. In Norway, within a week, our group developed a test kit to quickly isolate viral RNA and safely detect SARS-CoV-2 infection with sensitivity comparable to available kits. Herein, the procedure employed for the detection of SARS-CoV-2 in swab samples from patients using the NTNU-COVID-19 test kit is described in detail. This procedure, based on NAxtra magnetic nanoparticles and an optimized nucleic acid extraction procedure, is robust, reliable, and straightforward, providing high-quality nucleic acids within 14 min. The NAxtra protocol is adaptable and was further validated for extraction of DNA and RNA from other types of viruses. A comparison of the protocol on different liquid handling systems is also presented. Due to the simplicity and low cost of this method, implementation of this technology to diagnose virus infections on a clinical setting would benefit health care systems, promoting sustainability.
Assuntos
COVID-19 , Nanopartículas de Magnetita , Ácidos Nucleicos , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Teste para COVID-19 , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs-SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1-for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.
Assuntos
Ciclo Celular/genética , Proliferação de Células/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Técnicas de Silenciamento de Genes , Células HaCaT , Humanos , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , RNA-SeqRESUMO
BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.
Assuntos
Citosina , Ribossomos , Adenosina/análogos & derivados , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Animais , Citosina/análogos & derivados , DNA Helicases , Humanos , RNA Helicases , RNA Mensageiro/genética , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.
Assuntos
Antivirais/administração & dosagem , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Células A549 , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos/farmacologia , Antivirais/farmacologia , COVID-19 , Linhagem Celular , Infecções por Coronavirus/virologia , Bases de Dados de Produtos Farmacêuticos , Combinação de Medicamentos , Descoberta de Drogas , Sinergismo Farmacológico , Enterovirus Humano B/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Humanos , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Testes de Neutralização/métodos , Pneumonia Viral/tratamento farmacológico , Amodiaquina/farmacologia , Animais , COVID-19 , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/terapia , Quimioterapia Combinada , Emetina/farmacologia , Células HEK293 , Células HT29 , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Soros Imunes/imunologia , Imunização Passiva/métodos , Indóis , Nelfinavir/farmacologia , Pandemias , Piranos/farmacologia , Pirróis/farmacologia , SARS-CoV-2 , Células Vero , Soroterapia para COVID-19RESUMO
Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A-/-) develop fatty liver. RNA sequencing of male Oxr1A-/- liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A-/- pituitary gland and in rat Oxr1A-/- pituitary adenoma cell-line GH3. Oxr1A-/- male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland.
Assuntos
Arginina/metabolismo , Histonas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Feminino , Regulação da Expressão Gênica , Hormônio do Crescimento/sangue , Hormônio do Crescimento/metabolismo , Hormônios/metabolismo , Imunidade/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Especificidade de Órgãos , Hipófise/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , Ratos , Receptores da Somatotropina/metabolismo , Fator de Transcrição STAT5/metabolismo , Relação Estrutura-Atividade , Transcriptoma/genéticaRESUMO
Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3'end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression.
Assuntos
Cromatina/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Regulação da Expressão Gênica/genética , RNA Polimerase II/metabolismo , Cromatina/genética , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Expressão Gênica , Instabilidade Genômica , Células HEK293 , Humanos , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.
Assuntos
DNA Glicosilases/genética , Reparo do DNA/genética , Switching de Imunoglobulina/genética , Recombinação Genética/genética , Uracila-DNA Glicosidase/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Núcleo Celular/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Técnicas de Inativação de Genes , Genoma/genética , Humanos , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Isoformas de Proteínas/genética , Uracila/metabolismoRESUMO
To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double-strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom-generated and commercially available NHEJ-deficient human haploid HAP1 cells, PAXXΔ, XRCC4Δ , and XLFΔ . In our studies, HAP1 PAXXΔ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild-type controls. By contrast, XRCC4Δ and XLFΔ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double-strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx-/- and Aid-/- murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild-type levels in the Paxx-/- cells and completely ablated in the absence of activation-induced cytidine deaminase (AID). In addition, Paxx-/- CH12F3 cells were hypersensitive to zeocin when compared to wild-type controls. We concluded that Paxx-deficient mammalian cells maintain robust NHEJ and CSR.
RESUMO
Maintenance of a genome requires DNA repair integrated with chromatin remodeling. We have analyzed six transcriptome data sets and one data set on translational regulation of known DNA repair and remodeling genes in synchronized human cells. These data are available through our new database: www.dnarepairgenes.com. Genes that have similar transcription profiles in at least two of our data sets generally agree well with known protein profiles. In brief, long patch base excision repair (BER) is enriched for S phase genes, whereas short patch BER uses genes essentially equally expressed in all cell cycle phases. Furthermore, most genes related to DNA mismatch repair, Fanconi anemia and homologous recombination have their highest expression in the S phase. In contrast, genes specific for direct repair, nucleotide excision repair, as well as non-homologous end joining do not show cell cycle-related expression. Cell cycle regulated chromatin remodeling genes were most frequently confined to G1/S and S. These include e.g. genes for chromatin assembly factor 1 (CAF-1) major subunits CHAF1A and CHAF1B; the putative helicases HELLS and ATAD2 that both co-activate E2F transcription factors central in G1/S-transition and recruit DNA repair and chromatin-modifying proteins and DNA double strand break repair proteins; and RAD54L and RAD54B involved in double strand break repair. TOP2A was consistently most highly expressed in G2, but also expressed in late S phase, supporting a role in regulating entry into mitosis. Translational regulation complements transcriptional regulation and appears to be a relatively common cell cycle regulatory mechanism for DNA repair genes. Our results identify cell cycle phases in which different pathways have highest activity, and demonstrate that periodically expressed genes in a pathway are frequently co-expressed. Furthermore, the data suggest that S phase expression and over-expression of some multifunctional chromatin remodeling proteins may set up feedback loops driving cancer cell proliferation.
Assuntos
Ciclo Celular , Montagem e Desmontagem da Cromatina/genética , Reparo do DNA/genética , Expressão Gênica , Montagem e Desmontagem da Cromatina/fisiologia , Reparo do DNA/fisiologia , HumanosRESUMO
Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70-80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melfalan/farmacologia , Redes e Vias Metabólicas/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Estresse Oxidativo/genética , Transdução de Sinais/genética , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Humanos , Interleucina-8/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Proteoma/genética , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understanding the molecular pathways affected would add value to the interpretation of experimental observations subsequent to cell transfection. Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene®HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. The results were validated by western analysis as well as quantitative RT-PCR and this demonstrated that an essentially identical response was induced in HeLa by transfection using an empty pUC18 vector, which does not contain a mammalian virus promoter, as well as a liposome-based transfection reagent, Lipofectamine(TM)2000. Notably, no induction of the interferon response was observed in HEK293 cells, suggesting that these cells might be preferable to HeLa to avoid undesired off-target effects in transfection studies encompassing interferon-signaling and antiviral responses.
Assuntos
Plasmídeos/genética , Proteoma/metabolismo , Proteômica/métodos , Transfecção/métodos , Western Blotting , Isótopos de Carbono/metabolismo , Cromatografia Líquida/métodos , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Marcação por Isótopo/métodos , Lipídeos/química , Lisina/metabolismo , Proteoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem/métodosRESUMO
The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.
Assuntos
Citidina Desaminase/metabolismo , DNA de Neoplasias/metabolismo , Linfoma de Células B/genética , Mutação , Uracila/metabolismo , Pareamento Incorreto de Bases , Linhagem Celular Tumoral , Citosina/metabolismo , Reparo do DNA , Desaminação , Técnicas de Silenciamento de Genes , Humanos , Switching de Imunoglobulina , Linfoma de Células B/enzimologia , Linfoma de Células B/metabolismo , Mutação Puntual , Uracila-DNA Glicosidase/metabolismoRESUMO
Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies.
Assuntos
Reparo do DNA/genética , Linfoma de Células B/genética , Mutagênese , Uracila/metabolismo , Desaminase APOBEC-1 , Imunidade Adaptativa/genética , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citosina/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.
Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citidina Desaminase/imunologia , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Uracila-DNA Glicosidase/biossíntese , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calmodulina/metabolismo , Células Cultivadas , Reparo do DNA/genética , Proteínas de Ligação a DNA/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Uracila-DNA Glicosidase/genéticaRESUMO
Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Biomarcadores Tumorais/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Melfalan/farmacologia , Mieloma Múltiplo/genética , 8-Hidroxi-2'-Desoxiguanosina , Apoptose , Western Blotting , Ciclo Celular/genética , Proliferação de Células , Ensaio Cometa , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/genética , Proteína Quinase Ativada por DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Células Tumorais CultivadasRESUMO
Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 ß-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3.
Assuntos
Enzimas Reparadoras do DNA/química , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Dioxigenases/química , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Domínio Catalítico , DNA/química , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/química , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
It was recently reported that human immunodeficiency virus type 1 (HIV-1) Vpr induced the proteasomal degradation of the nuclear UNG2 enzyme for efficient virus replication. We confirm here that HIV-1 infection and Vpr expression reduce the level of endogenous UNG2, but this effect is not reverted by treatment with the proteasome inhibitor MG132. Moreover, this reduction is not mediated by Vpr binding to UNG2 and is independent of the Vpr-induced G(2) arrest. Finally, we show that Vpr influences the UNG2 promoter without affecting UNG1 gene expression. These data indicate that the Vpr-induced decrease of UNG2 level is mainly related to a transcriptional effect.
Assuntos
DNA Glicosilases/biossíntese , Regulação Viral da Expressão Gênica , Transcrição Gênica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Fase G2 , HIV-1/genética , Células HeLa , Humanos , Leupeptinas/farmacologia , Microscopia de Fluorescência/métodos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Replicação ViralRESUMO
The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.