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1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
J Virol ; 95(16): e0240120, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34076480

RESUMO

Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.


Assuntos
Farmacorresistência Viral/genética , Guanina/análogos & derivados , Vírus da Hepatite B/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/química , Inibidores da Transcriptase Reversa/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Guanina/metabolismo , Guanina/farmacologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Vírus da Hepatite B/química , Vírus da Hepatite B/enzimologia , Concentração Inibidora 50 , Cinética , Lamivudina/metabolismo , Lamivudina/farmacologia , Mutação , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Biosci Biotechnol Biochem ; 85(6): 1464-1467, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33826692

RESUMO

The α subunit of avian myeloblastosis virus reverse transcriptase (AMV-RT) is generated from the ß-subunit by proteolysis, and the αß heterodimer represents the active form. The codon-optimized gene was expressed in Escherichia coli, and an active αß heterodimer was generated. The RNA amplification activity of the purified recombinant AMV-RT αß heterodimer was similar to that of the native one.


Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Escherichia coli/genética , Multimerização Proteica , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Expressão Gênica , Estrutura Quaternária de Proteína , DNA Polimerase Dirigida por RNA/genética
4.
Methods Mol Biol ; 2263: 381-395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877609

RESUMO

Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS) enables determination of the molecular weight, oligomeric state, and stoichiometry of protein-nucleic acid complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a challenge in determination of molecular weight and stoichiometry based solely on the elution position from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the complex between the reverse transcriptase (RT) domain from group II intron-maturase from Eubacterium rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting repressor (FIR).


Assuntos
DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Cromatografia em Gel , DNA de Cadeia Simples/química , Eubacterium/genética , Eubacterium/metabolismo , Peso Molecular , Regiões Promotoras Genéticas , Multimerização Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Espalhamento de Radiação
5.
Sci Rep ; 11(1): 1820, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469065

RESUMO

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virologia , Temperatura Alta , Humanos , Nasofaringe/virologia , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Inativação de Vírus
6.
Dev Cell ; 56(1): 22-35.e7, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33278343

RESUMO

Retrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Meiose/genética , Proteínas de Ligação a RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a DNA/genética , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Meiose/fisiologia , Camundongos , Gambás/genética , Gambás/metabolismo , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/genética , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética
7.
Nucleic Acids Res ; 49(2): 1033-1045, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367793

RESUMO

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.


Assuntos
Adenina , Bacteriófagos/genética , Mutagênese , Retroelementos/genética , Adenina/química , Arginina/química , Sequência de Bases , Bordetella/virologia , Catálise , Sistema Livre de Células , Simulação por Computador , DNA Complementar/genética , Glicina/química , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Conformação Proteica , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/metabolismo
8.
Nat Commun ; 11(1): 5920, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219228

RESUMO

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Humanos , RNA/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/metabolismo , SARS-CoV-2 , Saliva/virologia , Vírion/genética
9.
J Biol Chem ; 295(50): 16975-16986, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33008888

RESUMO

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (-) SIVmac239-infected animals revealed that Vpx (-) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (-) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.


Assuntos
Mutação , Nucleotídeos/metabolismo , DNA Polimerase Dirigida por RNA/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/enzimologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Interações Hospedeiro-Patógeno , Cinética , Macaca mulatta , Macrófagos/metabolismo , Macrófagos/virologia , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/isolamento & purificação
10.
FEBS Lett ; 594(24): 4338-4356, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32970841

RESUMO

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Viral , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Sulfolobus , DNA Complementar/biossíntese , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática , Íons , Desnaturação Proteica , Domínios Proteicos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Temperatura
11.
Molecules ; 25(19)2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32992516

RESUMO

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5' terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Vírus da Hepatite B/fisiologia , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Tropolona/análogos & derivados , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Desaminase APOBEC-3G/metabolismo , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Tropolona/farmacologia
12.
Proc Natl Acad Sci U S A ; 117(35): 21274-21280, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817521

RESUMO

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.


Assuntos
Evolução Molecular , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA/biossíntese , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Conformação Proteica , DNA Polimerase Dirigida por RNA/química
13.
PLoS One ; 15(8): e0235102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857764

RESUMO

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.


Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Transcriptase Reversa do HIV/metabolismo , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Sequência de Bases , DNA Complementar/biossíntese , DNA Complementar/química , DNA Complementar/genética , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Humanos , Técnicas In Vitro , Inosina/análogos & derivados , Inosina/química , Inosina/metabolismo , Cinética , Camundongos , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Moldes Genéticos
14.
Genes (Basel) ; 11(8)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824672

RESUMO

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn2+ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m1A, m22G, m1G and m3C showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn2+ on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis.


Assuntos
Íons/metabolismo , Manganês/metabolismo , Transcrição Reversa , Pareamento de Bases , Íons/química , Manganês/química , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleosídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
15.
J Biol Chem ; 295(36): 12786-12795, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32699111

RESUMO

A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration-approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Encéfalo/metabolismo , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Recombinação Genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
16.
Nat Chem ; 12(8): 683-690, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690899

RESUMO

The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2'-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries D-altritol nucleic acid and 2'-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.


Assuntos
Evolução Molecular , DNA Polimerase Dirigida por RNA/metabolismo , RNA/metabolismo , Biblioteca Gênica , Vírus da Leucemia Murina/enzimologia , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , DNA Polimerase Dirigida por RNA/genética
17.
Anal Biochem ; 603: 113809, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32511965

RESUMO

Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease.


Assuntos
DNA/química , DNA/metabolismo , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA/biossíntese , Clivagem do DNA , Desoxirribonuclease I/metabolismo , Proteínas de Insetos , Mutação Puntual , Polimerização , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/metabolismo
19.
Chempluschem ; 85(5): 855-865, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32378814

RESUMO

We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus 1 (HIV-1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(DEA tC)TP as substrates. Michaelis-Menten kinetics for the insertion of these analogues show Vmax /KM from 0.0-5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV-1-RT, including RNA-dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(DEA tC)TP are compatible with a complete reverse transcription cycle in vitro.


Assuntos
Citidina/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/enzimologia , Citidina/análogos & derivados , HIV-1/enzimologia , Humanos , Cinética , Vírus da Leucemia Murina de Moloney/enzimologia , Especificidade por Substrato
20.
Anal Biochem ; 601: 113768, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32416095

RESUMO

Understanding reverse transcriptase (RT) activity is critical for designing fast one-step RT-PCRs. We report a stopped-flow assay that monitors SYBR Green I fluorescence to investigate RT activity in PCR conditions. We studied the influence of PCR conditions on RT activity and assessed the accuracy of cDNA synthesis predictions for one-step RT-PCR. Nucleotide incorporation increased from 26 to 89 s-1 between 1.5 and 6 mM MgCl2 but was largely unaffected by changes in KCl. Conversely, increasing KCl from 15 to 75 mM increased apparent rate constants for RT-oligonucleotide binding (0.010-0.026 nM-1 s-1) and unbinding (0.2-1.5 s-1). All rate constants increased between 22 and 42 °C. When evaluated by PCR quantification cycle, cDNA predictions differed from experiments using RNase H+ RT (average 1.7 cycles) and RNase H- (average 4.5 cycles). Decreasing H+ RT concentrations 10 to 104-fold from manufacturer recommendations improved cDNA predictions (average 0.8 cycles) and increased RT-PCR assay efficiency. RT activity assays and models can be used to aid assay design and improve the speed of RT-PCRs. RT type and concentration must be selected to promote rapid cDNA synthesis but minimize nonspecific amplification. We demonstrate 2-min one-step RT-PCR of a Zika virus target using reduced RT concentrations and extreme PCR.


Assuntos
DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fluorescência , Humanos , Cinética , Compostos Orgânicos/química
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