RESUMO
Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adenosina Desaminase/genética , Interferon Tipo I/imunologia , RNA de Cadeia Dupla/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Inosina/genética , Inosina/metabolismo , Interferon Tipo I/genética , Camundongos , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Edição de RNA/genética , RNA de Cadeia Dupla/metabolismoRESUMO
The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.
Assuntos
Replicação do DNA , Embrião de Mamíferos/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Animais , Instabilidade Cromossômica , DNA Polimerase Dirigida por DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Varicella-Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co-localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell-free system and phase-separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS.
Assuntos
Herpesvirus Humano 3 , Interferon Tipo I , Nucleotidiltransferases , Proteínas Virais , DNA/metabolismo , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/imunologia , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Proteínas de Membrana/imunologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/imunologia , Proteínas Virais/imunologiaRESUMO
Toll-like receptor 7 (TLR7) is an innate immune sensor for single-strand RNA (ssRNA). Recent structural analysis revealed that TLR7 has an additional binding site for nucleosides such as guanosine, and is activated when both guanosine and ssRNA bind. The nucleoside binding site also accommodates imidazoquinoline derivatives such as R848, which activate TLR7 in the absence of ssRNA. Here, we report that deoxyguanosine (dG) triggered cytokine production in murine bone marrow derived macrophages and plasmacytoid dendritic cells, as well as in human peripheral blood mononuclear cells, including type I interferons and pro-inflammatory factors such as TNF and IL-6. This signalling activity of dG was dependent on TLR7 and its adaptor MyD88 and did not require amplification via the type I interferon receptor. dG-triggered cytokine production required endosomal maturation but did not depend on the concurrent provision of RNA. We conclude that dG induces an inflammatory response through TLR7 and propose that dG is an RNA-independent TLR7 agonist.
Assuntos
Desoxiguanosina/imunologia , Inflamação/imunologia , Receptor 7 Toll-Like/agonistas , Animais , Desoxiguanosina/metabolismo , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.
Assuntos
Ácidos Graxos/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 2/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Herpes Simples/genética , Herpes Simples/patologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Lipoilação , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Células RAW 264.7RESUMO
Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Imunidade Inata/genética , Proteínas de Membrana/imunologia , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/genética , Nucleotidiltransferases/imunologia , Ribonuclease H/genética , Ribonucleases/genética , Animais , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Autoimunidade/genética , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ribonuclease H/metabolismoRESUMO
Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG-I-like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG-I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.
Assuntos
Proteína DEAD-box 58/imunologia , Interferon Tipo I/metabolismo , RNA/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Brasil , Proteína DEAD-box 58/genética , Regulação para Baixo , Células HEK293 , Humanos , Interferon Tipo I/genética , Fosforilação , Receptores Imunológicos , Transdução de Sinais , Replicação Viral , Zika virus , Infecção por Zika virusRESUMO
The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.
Assuntos
Células Dendríticas/metabolismo , Imunidade Inata/imunologia , Modelos Imunológicos , Ácidos Nucleicos Heteroduplexes/metabolismo , Transdução de Sinais/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Western Blotting , Células Dendríticas/imunologia , Endossomos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Ácidos Nucleicos Heteroduplexes/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Receptor Toll-Like 9/imunologiaRESUMO
Post-transcriptional control determines the fate of cellular RNA molecules. Nonsense-mediated decay (NMD) provides quality control of mRNA, targeting faulty cellular transcripts for degradation by multiple nucleases including the RNA exosome. Recent findings have revealed a role for NMD in targeting viral RNA molecules, thereby restricting virus infection. Interestingly, NMD is also linked to immune responses at another level: mutations affecting the NMD or RNA exosome machineries cause chronic activation of defence programmes, resulting in autoimmune phenotypes. Here we place these observations in the context of other links between innate antiviral immunity and type I interferon mediated disease and examine two models: one in which expression or function of pathogen sensors is perturbed and one wherein host-derived RNA molecules with a propensity to activate such sensors accumulate.
Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , RNA Viral/imunologia , Vírus/imunologia , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/patologia , Autoimunidade , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/patologia , RNA Viral/metabolismo , Transdução de SinaisRESUMO
SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1(-/-) cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism.
Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Malformações do Sistema Nervoso/metabolismo , Transcrição Reversa/fisiologia , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Linhagem Celular , Vetores Genéticos/genética , Infecções por HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Interferon Tipo I/metabolismo , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/genética , Malformações do Sistema Nervoso/genética , Nucleotídeos/metabolismo , Transcrição Reversa/genética , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Although we can now measure single-cell signaling responses with multivariate, high-throughput techniques our ability to interpret such measurements is still limited. Even interpretation of dose-response based on single-cell data is not straightforward: signaling responses can differ significantly between cells, encompass multiple signaling effectors, and have dynamic character. Here, we use probabilistic modeling and information-theory to introduce fractional response analysis (FRA), which quantifies changes in fractions of cells with given response levels. FRA can be universally performed for heterogeneous, multivariate, and dynamic measurements and, as we demonstrate, quantifies otherwise hidden patterns in single-cell data. In particular, we show that fractional responses to type I interferon in human peripheral blood mononuclear cells are very similar across different cell types, despite significant differences in mean or median responses and degrees of cell-to-cell heterogeneity. Further, we demonstrate that fractional responses to cytokines scale linearly with the log of the cytokine dose, which uncovers that heterogeneous cellular populations are sensitive to fold-changes in the dose, as opposed to additive changes.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/metabolismo , Modelos Imunológicos , Células 3T3 , Animais , Voluntários Saudáveis , Humanos , Interferon Tipo I/imunologia , Leucócitos Mononucleares/imunologia , Camundongos , Modelos Estatísticos , Cultura Primária de Células , Transdução de Sinais/imunologia , Análise de Célula Única , SoftwareRESUMO
Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.
Assuntos
Fibrose Pulmonar Idiopática/imunologia , Interferon Tipo I/metabolismo , Pulmão/imunologia , Monócitos/imunologia , Estudos de Casos e Controles , Células Cultivadas , Quimiocina CCL2/sangue , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Imunofenotipagem , Interferon Tipo I/genética , Interleucina-6/sangue , Pulmão/metabolismo , Pulmão/patologia , Fator Estimulador de Colônias de Macrófagos/sangue , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/metabolismo , Fenótipo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Análise de Célula ÚnicaRESUMO
Enzymes that degrade nucleic acids are emerging as important players in the pathogenesis of inflammatory disease. This is exemplified by the recent identification of four genes that cause the childhood inflammatory disorder, Aicardi-Goutières syndrome (AGS). This is an autosomal recessive neurological condition whose clinical and immunological features parallel those of congenital viral infection. The four AGS genes encode two nucleases: TREX1 and the hetero-trimeric Ribonuclease H2 (RNase H2) complex. The biochemical activity of these enzymes was initially characterised 30 years ago but a role in neurological inflammation was entirely unanticipated until they were found to be mutated in AGS. This has led to a hypothesis that accumulation of intracellular nucleic acids occurs as a consequence of mutation in these enzymes and triggers an inflammatory response through activation of innate immune pattern recognition receptors.
Assuntos
Imunidade Inata/fisiologia , Inflamação/metabolismo , Ácidos Nucleicos/imunologia , Autoimunidade/genética , Encéfalo/patologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Inflamação/genética , Mutação , Ácidos Nucleicos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , SíndromeRESUMO
The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.
Assuntos
Nucleotídeos de Desoxiguanina/metabolismo , Nucleosídeos de Purina/farmacologia , Pirimidinonas/farmacologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Vírus da Influenza A/fisiologia , Interferon Tipo I/metabolismo , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Imunidade Inata , Vírus da Influenza A/metabolismo , Interferon Tipo I/genética , Pulmão/metabolismo , Pulmão/virologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Proteínas não Estruturais Virais/deficiência , Proteínas não Estruturais Virais/metabolismoRESUMO
We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.
Assuntos
Replicação do DNA , DNA Mitocondrial/química , Animais , DNA , Humanos , Mamíferos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNARESUMO
This study describes a new tool for accurate and reliable high-throughput detection of copy number variation in the human genome. We have constructed a large-insert clone DNA microarray covering the entire human genome in tiling path resolution that we have used to identify copy number variation in human populations. Crucial to this study has been the development of a robust array platform and analytic process for the automated identification of copy number variants (CNVs). The array consists of 26,574 clones covering 93.7% of euchromatic regions. Clones were selected primarily from the published "Golden Path," and mapping was confirmed by fingerprinting and BAC-end sequencing. Array performance was extensively tested by a series of validation assays. These included determining the hybridization characteristics of each individual clone on the array by chromosome-specific add-in experiments. Estimation of data reproducibility and false-positive/negative rates was carried out using self-self hybridizations, replicate experiments, and independent validations of CNVs. Based on these studies, we developed a variance-based automatic copy number detection analysis process (CNVfinder) and have demonstrated its robustness by comparison with the SW-ARRAY method.