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1.
Mol Cell ; 79(6): 1037-1050.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32882183

RESUMO

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.


Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto Jovem
2.
Nucleic Acids Res ; 52(3): 1207-1225, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38117983

RESUMO

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.


Assuntos
Replicação do DNA , Genoma Mitocondrial , Ribonucleosídeos , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Ribonucleosídeos/metabolismo , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo
3.
J Biol Chem ; 295(46): 15438-15453, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32883809

RESUMO

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Kit de Reagentes para Diagnóstico/economia , SARS-CoV-2/genética , Transferência de Tecnologia , Universidades/economia , Biotecnologia/métodos , COVID-19/virologia , Humanos , Kit de Reagentes para Diagnóstico/provisão & distribuição , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/isolamento & purificação
4.
J Biol Chem ; 294(35): 13061-13072, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31300556

RESUMO

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.


Assuntos
DNA/metabolismo , Escherichia coli/metabolismo , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Ribosemonofosfatos/metabolismo , Células HeLa , Humanos , Oxirredução , Células Tumorais Cultivadas
5.
Chembiochem ; 21(23): 3359-3370, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32705742

RESUMO

Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3' phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.


Assuntos
Carbodi-Imidas/química , DNA/química , Temperatura
6.
EMBO J ; 34(18): 2363-82, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26303906

RESUMO

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.


Assuntos
Amiloide/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Organelas/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Amiloide/genética , Proteínas de Transporte/genética , DNA Helicases/genética , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organelas/genética , Organelas/patologia
7.
J Biol Chem ; 292(49): 20113-20124, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29038292

RESUMO

Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [PSI+] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [PSI+] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (pep4Δ) or PrB (prb1Δ), increased the rate of de novo formation of [PSI+] prion up to ∼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [PSI+] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [PSI+] strains nor eliminated preexisting [PSI+]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [PSI+] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.


Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Príons/antagonistas & inibidores , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeo Hidrolases/metabolismo , Príons/metabolismo , Vacúolos/enzimologia , Leveduras/metabolismo
8.
Mol Microbiol ; 105(2): 242-257, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28431189

RESUMO

Yeast self-perpetuating protein aggregates (prions) provide a convenient model for studying various components of the cellular protein quality control system. Molecular chaperones and chaperone-sorting factors, such as yeast Cur1 protein, play key role in proteostasis via tight control of partitioning and recycling of misfolded proteins. In this study, we show that, despite the previously described ability of Cur1 to antagonize the yeast prion [URE3], it enhances propagation and phenotypic manifestation of another prion, [PSI+ ]. We demonstrate that both curing of [URE3] and enhancement of [PSI+ ] in the presence of excess Cur1 are counteracted by the cochaperone Hsp40-Sis1 in a dosage-dependent manner, and show that the effect of Cur1 on prions parallels effects of the attachment of nuclear localization signal to Sis1, indicating that Cur1 acts on prions via its previously reported ability to relocalize Sis1 from the cytoplasm to nucleus. This shows that the direction in which Cur1 influences a prion depends on how this specific prion responds to relocalization of Sis1.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/metabolismo , Sinais de Localização Nuclear/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismo , Dobramento de Proteína , Transporte Proteico , Saccharomyces cerevisiae/metabolismo
9.
Mol Microbiol ; 96(3): 621-32, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25649498

RESUMO

Cross-beta fibrous protein aggregates (amyloids and amyloid-based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI(+)], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome-associated complex (RAC), composed of Hsp40-Zuo1 and Hsp70-Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC-deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed because of lack of ribosome-associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild-type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb-based regulatory circuit.


Assuntos
Retroalimentação , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/biossíntese , Príons/biossíntese , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo
10.
J Biol Chem ; 289(40): 27625-39, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25143386

RESUMO

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Resposta ao Choque Térmico , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Fatores de Terminação de Peptídeos/genética , Príons/genética , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
PLoS Genet ; 8(4): e1002634, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22536159

RESUMO

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.


Assuntos
Dosagem de Genes , Fatores de Terminação de Peptídeos , Peptídeos , Príons/genética , Proteínas de Saccharomyces cerevisiae , Epigênese Genética , Humanos , Proteína Huntingtina , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/toxicidade , Príons/química , Príons/metabolismo , Ligação Proteica/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
12.
iScience ; 27(6): 110012, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38868188

RESUMO

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered orthologs of the human RNASEH2A-G37S and RNASEH2C-R69W AGS mutations in yeast Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these AGS-ortholog mutants. We found a high rNMP presence in the nuclear genome of rnh201-G42S-mutant cells, and an elevated rCMP content in both mutants, reflecting preferential cleavage of RNase H2 at rGMP. We discovered unique rNMP patterns in each mutant, showing differential activity of the AGS mutants on the leading or lagging replication strands. This study guides future research on rNMP characteristics in human genomes with AGS mutations.

13.
bioRxiv ; 2023 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-37873120

RESUMO

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS-ortholog mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these yeast AGS-ortholog mutants. We found higher rNMP incorporation in the nuclear genome of rnh201-G42S than in wild-type and rnh203-K46W-mutant cells, and an elevated rCMP content in both mutants. Moreover, we uncovered unique rNMP patterns in each mutant, highlighting a differential activity of the AGS mutants towards rNMPs embedded on the leading or on the lagging strand of DNA replication. This study guides future research on rNMP characteristics in human genomic samples carrying AGS mutations.

14.
Mol Microbiol ; 76(6): 1483-99, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20444092

RESUMO

Self-perpetuating amyloid-based protein isoforms (prions) transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Although mechanisms that control species specificity of prion transmission are poorly understood, studies of closely related orthologues of yeast prion protein Sup35 demonstrate that cross-species prion transmission is modulated by both genetic (specific sequence elements) and epigenetic (prion variants, or 'strains') factors. Depending on the prion variant, the species barrier could be controlled at the level of either heterologous co-aggregation or conversion of the aggregate-associated heterologous protein into a prion polymer. Sequence divergence influences cross-species transmission of different prion variants in opposing ways. The ability of a heterologous prion domain to either faithfully reproduce or irreversibly switch the variant-specific prion patterns depends on both sequence divergence and the prion variant. Sequence variations within different modules of prion domains contribute to transmission barriers in different cross-species combinations. Individual amino acid substitutions within short amyloidogenic stretches drastically alter patterns of cross-species prion conversion, implicating these stretches as major determinants of species specificity.


Assuntos
Regulação Fúngica da Expressão Gênica , Transferência Genética Horizontal , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Polimorfismo Genético , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Amiloide/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência
15.
Chem Commun (Camb) ; 57(99): 13602-13605, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34852364

RESUMO

A goal of supramolecular chemistry is to create covalent polymers of precise composition and stereochemistry from complex mixtures by the reversible assembly of specific monomers prior to covalent bond formation. We illustrate the power of this approach with short oligomers of deoxyadenosine monophosphate ((dA)n3'p), n ≥ 3, which form supramolecular assemblies with cyanuric acid. The addition of a condensing agent to these assemblies results in their selective, non-enzymatic polymerization to form long polymers (e.g., (dA)1003'p). Significantly, mixtures of D- and L-(dA)53'p form homochiral covalent polymers, which demonstrates self-sorting of racemic monomers and covalent bond formation exclusively in homochiral assemblies.


Assuntos
Oligonucleotídeos/química , Configuração de Carboidratos , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Polimerização , Estereoisomerismo
16.
Diagnostics (Basel) ; 11(5)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069462

RESUMO

OBJECTIVES: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. METHODS: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. RESULTS: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60-180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. CONCLUSION: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

17.
iScience ; 24(1): 102005, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33490913

RESUMO

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genome of cells. The distribution of rNMPs in DNA has been studied only in limited genomes. Using the ribose-seq protocol and the Ribose-Map bioinformatics toolkit, we reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. We discovered a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the corresponding nucleotide content of these C. reinhardtii organelle genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. We reveal specific biases and patterns in rNMP distributions in the algal mitochondrial, chloroplast, and nuclear DNA. Moreover, we identified the C. reinhardtii orthologous genes for all three subunits of the RNase H2 enzyme using GeneMark-EP + gene finder.

18.
Nat Commun ; 11(1): 2447, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415081

RESUMO

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.


Assuntos
Citosina/metabolismo , DNA Fúngico/genética , Desoxiadenosinas/metabolismo , Genoma Fúngico , Guanosina/metabolismo , Ribonucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Núcleo Celular/genética , DNA Mitocondrial/genética , Genoma Mitocondrial , Sequências Repetitivas de Ácido Nucleico/genética , Ribonuclease H/metabolismo , Schizosaccharomyces/genética
19.
medRxiv ; 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32766604

RESUMO

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

20.
J Cell Biol ; 157(6): 997-1004, 2002 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-12058016

RESUMO

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas do Tecido Nervoso/toxicidade , Proteínas Nucleares/toxicidade , Ácido Poliglutâmico/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cruzamentos Genéticos , Meios de Cultura , Proteínas Fúngicas/genética , Deleção de Genes , Frequência do Gene , Genótipo , Proteínas de Fluorescência Verde , Doença de Huntington/genética , Proteínas Luminescentes , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Príons/genética , Regiões Promotoras Genéticas , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/fisiologia , Solubilidade , Supressão Genética
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