RESUMO
Survival and genome stability are critical characteristics of healthy cells. DNA palindromes pose a threat to genome stability and have been shown to participate in a reaction leading to the formation of inverted chromosome duplications centered around themselves. There is considerable interest in the mechanism of this rearrangement given its likely contribution to genome instability in cancer cells. This study shows that formation of large inverted chromosome duplications can be observed in the chromosome of Escherichia coli. They are formed at the site of a 246 bp interrupted DNA palindrome in the absence of the hairpin nuclease SbcCD and the recombination protein RecA. The genetic requirements for this spontaneous rearrangement are consistent with a pathway involving DNA degradation and hairpin formation, as opposed to a cruciform cleavage pathway. Accordingly, the formation of palindrome-dependent hairpin intermediates can be induced by an adjacent DNA double-stand break.
Assuntos
Cromossomos Bacterianos/metabolismo , Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Exonucleases/metabolismo , Rearranjo Gênico , Sequências Repetidas Invertidas/genética , Recombinases Rec A/metabolismo , Pareamento de Bases/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/genética , Microscopia , Modelos Biológicos , Recombinação Genética/genéticaRESUMO
Long DNA palindromes are sites of genome instability (deletions, amplification, and translocations) in both prokaryotic and eukaryotic cells. In Escherichia coli, genetic evidence has suggested that they are sites of DNA cleavage by the SbcCD complex that can be repaired by homologous recombination. Here we obtain in vivo physical evidence of an SbcCD-induced DNA double-strand break (DSB) at a palindromic sequence in the E. coli chromosome and show that both ends of the break stimulate recombination. Cleavage is dependent on DNA replication, but the observation of two ends at the break argues that cleavage does not occur at the replication fork. Genetic analysis shows repair of the break requires the RecBCD recombination pathway and PriA, suggesting a mechanism of bacterial DNA DSB repair involving the establishment of replication forks.
Assuntos
Sequência de Bases , Cromossomos Bacterianos , Quebras de DNA de Cadeia Dupla , Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Exonucleases/metabolismo , Dano ao DNA , Reparo do DNA , Desoxirribonucleases/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Exodesoxirribonuclease V/metabolismo , Exonucleases/genética , Recombinação GenéticaRESUMO
Helicase-nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Desoxirribonucleases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sequência Conservada , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/metabolismo , Desoxirribonucleases/metabolismo , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Ribonuclease H/química , Sulfolobus solfataricus/enzimologiaRESUMO
A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.
Assuntos
Acridinas/química , Imunoensaio/métodos , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Animais , Anticorpos Imobilizados/química , AMP Cíclico/análise , Peroxidase do Rábano Silvestre/análise , Humanos , Interleucina-8/análise , Camundongos , Antígeno Prostático Específico/análise , Ratos , OvinosRESUMO
During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5'-3' end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.
Assuntos
Dano ao DNA , DNA Arqueal/genética , DNA Bacteriano/genética , DNA/genética , Células Eucarióticas/metabolismo , FilogeniaRESUMO
Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR.
Assuntos
Cromossomos Bacterianos/genética , Reparo do DNA , Escherichia coli/genética , Instabilidade de Microssatélites , Sequências de Repetição em Tandem/genética , Repetições de Trinucleotídeos/genética , Sequência de Bases , Quebras de DNA de Cadeia Dupla , DNA Bacteriano/genética , Humanos , Modelos Genéticos , Recombinação Genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.
Assuntos
Linhagem da Célula , Células Epiteliais/citologia , Próstata/citologia , Células-Tronco/citologia , Células Clonais , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , MicrodissecçãoRESUMO
As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here, we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (B.1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.
RESUMO
Goal: Monitoring the genetic diversity and emerging mutations of SARS-CoV-2 is crucial for understanding the evolution of the virus and assuring the performance of diagnostic tests, vaccines, and therapies against COVID-19. SARS-CoV-2 is still adapting to humans and, as illustrated by B.1.1.7 (Alpha) and B.1.617.2 (Delta), lineage dynamics are fluid, and strain prevalence may change radically in a matter of months. The National Institutes of Health's Rapid Acceleration of Diagnostics (RADxSM) initiative created a Variant Task Force to assess the impact of emerging SARS-CoV-2 variants on in vitro diagnostic testing. Working in tandem with clinical laboratories, the FDA, and the CDC, the Variant Task Force uses both in silico modeling and in vitro testing to determine the effect of SARS-CoV-2 mutations on diagnostic molecular and antigen tests. Here, we offer an overview of the approach and activities of the RADx Variant Task Force to ensure test performance against emerging SARS-CoV-2 lineages.
RESUMO
Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.
Assuntos
DNA Mitocondrial/genética , Testes Genéticos/métodos , Doenças Mitocondriais/diagnóstico , Urotélio , Análise Mutacional de DNA , Fluorescência , Aconselhamento Genético , Humanos , Doenças Mitocondriais/genética , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Deleção de SequênciaRESUMO
Expanded CAG x CTG trinucleotide repeat tracts are associated with several human inherited diseases, including Huntington's disease, myotonic dystrophy, and spinocerebellar ataxias. Here we describe a new model system to investigate repeat instability in the Escherichia coli chromosome. Using this system, we reveal patterns of deletion instability consistent with secondary structure formation in vivo and address the molecular basis of orientation-dependent instability. We demonstrate that the orientation dependence of CAG x CTG trinucleotide repeat deletion is determined by the proofreading subunit of DNA polymerase III (DnaQ) in the presence of the hairpin nuclease SbcCD (Rad50/Mre11). Our results suggest that, although initiation of slippage can occur independently of CAG x CTG orientation, the folding of the intermediate affects its processing and this results in orientation dependence. We propose that proofreading is inefficient on the CTG-containing strand because of its ability to misfold and that SbcCD contributes to processing in a manner that is dependent on proofreading and repeat tract orientation. Furthermore, we demonstrate that transcription and recombination do not influence instability in this system.
Assuntos
Instabilidade Cromossômica/genética , DNA Bacteriano/genética , Escherichia coli/genética , Conformação de Ácido Nucleico , Expansão das Repetições de Trinucleotídeos/genética , Sequência de Bases , Cromossomos Bacterianos/genética , DNA Polimerase III/genética , Replicação do DNA , DNA Bacteriano/biossíntese , DNA Bacteriano/química , Proteínas de Escherichia coli/genética , Exonucleases , Modelos Genéticos , Dados de Sequência Molecular , Recombinação Genética , Deleção de Sequência/genética , Transcrição GênicaAssuntos
Indústria Farmacêutica/legislação & jurisprudência , Regulamentação Governamental , Cardiopatias/diagnóstico , Troponina I/sangue , Troponina T/sangue , Síndrome Coronariana Aguda/diagnóstico , Biomarcadores/sangue , Ensaios Clínicos como Assunto/legislação & jurisprudência , Humanos , Infarto do Miocárdio/diagnóstico , Kit de Reagentes para Diagnóstico , Valores de Referência , Sensibilidade e Especificidade , Estados Unidos , United States Food and Drug AdministrationRESUMO
In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.
Assuntos
Proteínas Arqueais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Sulfolobus acidocaldarius/genética , Ubiquitinas/genética , Proteínas Arqueais/metabolismo , Cromatografia em Gel , Cromatografia Líquida , Dicroísmo Circular , Cristalografia por Raios X , Espectrometria de Massas , Microscopia Eletrônica , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Proteólise , Sulfolobus acidocaldarius/metabolismo , Ubiquitinas/metabolismoRESUMO
The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Archaea/enzimologia , Proteínas Arqueais/metabolismo , DNA Arqueal/genética , Translocação Genética , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Quebras de DNA de Cadeia Dupla , DNA Arqueal/metabolismo , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The ability to detect mitochondrial DNA (mtDNA) variation within human cells is important not only to identify mutations causing mtDNA disease, but also as mtDNA mutations are being increasingly described in many ageing tissues and in complex diseases such as diabetes, neurodegeneration and cancer. In this review, we discuss the main molecular genetic techniques that can be applied to study the two main types of mtDNA mutation: point mutations and large-scale mtDNA rearrangements. We then describe in detail protocols routinely used within our laboratory to analyse mtDNA mutations in individual human cells such as single muscle fibres and individual neurons to study the relationship between mtDNA mutation load and respiratory chain dysfunction.
Assuntos
DNA Mitocondrial/análise , Técnicas Genéticas , Envelhecimento/genética , Humanos , Mitocôndrias/genética , Mutação PuntualRESUMO
Mitochondrial DNA (mtDNA) deletions are a primary cause of mitochondrial disease and are likely to have a central role in the aging of postmitotic tissues. Understanding the mechanism of the formation and subsequent clonal expansion of these mtDNA deletions is an essential first step in trying to prevent their occurrence. We review the previous literature and recent results from our own laboratories, and conclude that mtDNA deletions are most likely to occur during repair of damaged mtDNA rather than during replication. This conclusion has important implications for prevention of mtDNA disease and, potentially, for our understanding of the aging process.