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1.
J Transl Med ; 19(1): 287, 2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34217309

RESUMO

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 Ligases
2.
Nucleic Acids Res ; 47(9): 4569-4585, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30838409

RESUMO

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/metabolismo
3.
Biochim Biophys Acta ; 1854(1): 84-90, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25448019

RESUMO

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/métodos
4.
Sci Rep ; 13(1): 11714, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474666

RESUMO

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 Especificidade
5.
Sci Rep ; 13(1): 20836, 2023 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-38012172

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/genética
6.
Nucleic Acids Res ; 38(19): 6447-55, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20525795

RESUMO

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 Substrato
7.
Sci Rep ; 11(1): 18952, 2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34556693

RESUMO

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-Seq
8.
DNA Repair (Amst) ; 8(7): 822-33, 2009 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-19411194

RESUMO

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.


Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-2/metabolismo , Uracila-DNA Glicosidase/genética , Sequência de Bases , Sítios de Ligação/genética , Fator de Ligação a CCAAT/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Fatores de Transcrição E2F/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição AP-2/genética , Transfecção , Tretinoína/farmacologia
9.
J Virol ; 83(19): 10256-63, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19625402

RESUMO

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 Viral
10.
Viruses ; 12(10)2020 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-33080984

RESUMO

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-19
11.
Viruses ; 12(6)2020 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545799

RESUMO

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-19
12.
Cell Rep ; 30(12): 4165-4178.e7, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209476

RESUMO

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/genética
13.
Nat Commun ; 10(1): 5460, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784530

RESUMO

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/metabolismo
14.
FEBS Open Bio ; 8(3): 442-448, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29511621

RESUMO

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.

15.
Nucleic Acids Res ; 32(11): 3456-61, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15229293

RESUMO

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Metilação de DNA , Enzimas Reparadoras do DNA , DNA de Cadeia Simples/metabolismo , Dioxigenases , Humanos , Magnésio/farmacologia , Metilação , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , RNA/metabolismo , RNA Complementar/química , RNA de Cadeia Dupla/metabolismo , Especificidade por Substrato
16.
Nucleic Acids Res ; 32(18): 5486-98, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15479784

RESUMO

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Divisão Celular , Linhagem Celular , DNA/química , DNA/metabolismo , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Células HeLa , Humanos , Substâncias Macromoleculares , Testes de Precipitina , Especificidade por Substrato , Uracila/metabolismo , Uracila-DNA Glicosidase , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
17.
DNA Repair (Amst) ; 3(11): 1389-407, 2004 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-15380096

RESUMO

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.


Assuntos
Dano ao DNA , Reparo do DNA , DNA/química , DNA/metabolismo , RNA/química , RNA/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase , Alquilantes/metabolismo , Alquilantes/toxicidade , Alquilação , Sequência de Aminoácidos , Animais , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos
18.
DNA Repair (Amst) ; 30: 53-67, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25881042

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 , Humanos
19.
DNA Repair (Amst) ; 25: 60-71, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25486549

RESUMO

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/metabolismo
20.
PLoS One ; 10(3): e0119857, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25769101

RESUMO

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/genética
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