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1.
Nat Biotechnol ; 39(9): 1129-1140, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504351

RESUMO

Assessing the reproducibility, accuracy and utility of massively parallel DNA sequencing platforms remains an ongoing challenge. Here the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Study benchmarks the performance of a set of sequencing instruments (HiSeq/NovaSeq/paired-end 2 × 250-bp chemistry, Ion S5/Proton, PacBio circular consensus sequencing (CCS), Oxford Nanopore Technologies PromethION/MinION, BGISEQ-500/MGISEQ-2000 and GS111) on human and bacterial reference DNA samples. Among short-read instruments, HiSeq 4000 and X10 provided the most consistent, highest genome coverage, while BGI/MGISEQ provided the lowest sequencing error rates. The long-read instrument PacBio CCS had the highest reference-based mapping rate and lowest non-mapping rate. The two long-read platforms PacBio CCS and PromethION/MinION showed the best sequence mapping in repeat-rich areas and across homopolymers. NovaSeq 6000 using 2 × 250-bp read chemistry was the most robust instrument for capturing known insertion/deletion events. This study serves as a benchmark for current genomics technologies, as well as a resource to inform experimental design and next-generation sequencing variant calling.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Pareamento Incorreto de Bases , Benchmarking , DNA/genética , DNA Bacteriano/genética , Genoma Bacteriano , Genoma Humano , Humanos
2.
J Microbiol Biotechnol ; 31(7): 903-911, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34261850

RESUMO

Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed, and future research and development directions are also proposed.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma Microbiano/genética , Pareamento Incorreto de Bases , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Motivos de Nucleotídeos , RNA Guia/química , RNA Guia/metabolismo
3.
Chem Commun (Camb) ; 57(65): 8039-8042, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34291259

RESUMO

Two-dimensional (2D) hexagonal boron nitride nanosheet (h-BNNS) is proposed as an effective nanoquencher for fluorescence detection of biocompatible microRNA. Compared with bulk hexagonal boron nitride (h-BN), the exfoliated ultrathin nanosheet has a narrow band gap and increased conductivity, thus enabling fast electron transfer with this electron acceptor for more effective fluorescence detection of microRNA. Remarkably, using the nanoprobe consisting of h-BNNS and FAM dye-labeled ssDNA, a low detection limit of 2.39 nM is achieved and a rapid fluorescence response is observed compared with previously reported fluorescence sensing materials. More importantly, this sensing system could also distinguish base-mismatched microRNA, suggesting that the proposed sensing platform held excellent selectivity and great promise for application in the detection of nucleotide polymorphism. This work will benefit microRNA-related fundamental research and disease diagnosis.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , MicroRNAs/análise , Nanoestruturas/química , Pareamento Incorreto de Bases , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Fluoresceínas/química , Fluorescência , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
4.
Nat Commun ; 12(1): 3759, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145298

RESUMO

Pol µ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol µ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol µ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol µ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol µ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.


Assuntos
Pareamento Incorreto de Bases/genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica/genética , Pareamento de Bases/genética , DNA/química , DNA Ligases/metabolismo , DNA Polimerase Dirigida por DNA/genética , Guanosina Trifosfato/química , Humanos
5.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063755

RESUMO

Energetically unfavorable Watson-Crick (WC)-like tautomeric forms of nucleobases are known to introduce spontaneous mutations, and contribute to replication, transcription, and translation errors. Recent NMR relaxation dispersion techniques were able to show that wobble (w) G•U mispair exists in equilibrium with the short-lived, low-population WC-like enolic tautomers. Presently, we have investigated the wG•U → WC-like enolic reaction pathway using various theoretical methods: quantum mechanics (QM), molecular dynamics (MD), and combined quantum mechanics/molecular mechanics (QM/MM). The previous studies on QM gas phase calculations were inconsistent with experimental data. We have also explored the environmental effects on the reaction energies by adding explicit water. While the QM-profile clearly becomes endoergic in the presence of water, the QM/MM-profile remains consistently endoergic in the presence and absence of water. Hence, by including microsolvation and QM/MM calculations, the experimental data can be explained. For the G•Uenol→ Genol•U pathway, the latter appears to be energetically more favorable throughout all computational models. This study can be considered as a benchmark of various computational models of wG•U to WC-like tautomerization pathways with and without the environmental effects, and may contribute on further studies of other mispairs as well.


Assuntos
Guanina/metabolismo , RNA/genética , Uracila/metabolismo , Pareamento Incorreto de Bases/genética , Pareamento de Bases/genética , Simulação por Computador , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação Puntual/genética , Teoria Quântica
6.
Cells ; 10(6)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070467

RESUMO

The DNA repair endonuclease EndoMS/NucS is highly conserved in Archaea and Actinobacteria. This enzyme is able to recognize and cleave dsDNA carrying a mismatched base pair, and its activity is enhanced by the interaction with the sliding clamp of the replisome. Today, EndoMS/NucS has been established as the key protein of a non-canonical mismatch repair (MMR) pathway, acting specifically in the repair of transitions and being essential for maintaining genome stability. Despite having some particularities, such as its lower activity on transversions and the inability to correct indels, EndoMS/NucS meets the main hallmarks of a MMR. Its absence leads to a hypermutator phenotype, a transition-biased mutational spectrum and an increase in homeologous recombination. Interestingly, polymorphic EndoMS/NucS variants with a possible effect in mutation rate have been detected in clinical isolates of the relevant actinobacterial pathogen Mycobacterium tuberculosis. Considering that MMR defects are often associated with the emergence of resistant bacteria, the existence of EndoMS/NucS-defective mutators could have an important role in the acquisition of antibiotic resistance in M. tuberculosis. Therefore, a further understanding of the EndoMS/NucS-mediated non-canonical MMR pathway may reveal new strategies to predict and fight drug resistance. This review is focused on the recent progress in NucS, with special emphasis on its effect on genome stability and evolvability in Actinobacteria.


Assuntos
Actinobacteria , Proteínas de Bactérias/metabolismo , Reparo de Erro de Pareamento de DNA , Actinobacteria/genética , Actinobacteria/metabolismo , Pareamento Incorreto de Bases , Instabilidade Genômica , Taxa de Mutação
7.
Nucleic Acids Res ; 49(9): 5278-5293, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34009379

RESUMO

The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/genética , Francisella/enzimologia , Mutação , Conformação Proteica
8.
Chemistry ; 27(41): 10574-10577, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34014006

RESUMO

Dodecamer duplex DNA containing anomeric (α/ß-d) and enantiomeric (ß-l/ß-d) 2'-deoxycytidine mismatches was studied with respect to base pair stability in the absence and presence of silver ions. Stable duplexes with silver-mediated cytosine-cytosine pairs were formed by all anomeric and enantiomeric combinations. Stability changes were observed depending on the composition of the mismatches. Most strikingly, the new silver-mediated base pair of anomeric α-d-dC with enantiomeric ß-l-dC is superior to the well-noted ß-d/ß-d-dC pair in terms of stability. CD spectra were used to follow global helical changes of DNA structure.


Assuntos
DNA , Prata , Pareamento Incorreto de Bases , Pareamento de Bases , Citosina , Íons , Conformação de Ácido Nucleico
9.
Nucleic Acids Res ; 49(9): 5084-5094, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33877329

RESUMO

DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPß binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPß binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.


Assuntos
Pareamento Incorreto de Bases , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/química , Citosina/química , DNA/química , DNA/metabolismo , Reparo do DNA , Guanina/química , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Timina/química
10.
Comput Biol Chem ; 92: 107485, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33872920

RESUMO

The seed of life is concealed in the base sequence in DNA. This macromolecule is continuously exposed to harmful factors which can cause it damage. The stability of genetic information depends on the protein efficiency of repair systems. Glycosylases are the scouts which recognize and remove damaged bases. Their efficiency depends on how rapidly they recognize DNA lesions. One theory states that charge transfer is involved in protein cross talking through ds-DNA. For these reasons a comparative analysis of ds-oligo containing a mismatched base pair dA:::dG and a damaged dA::dGOXO is proposed. Additionally, the electronic properties of the short ds-oligo in the context of non-equilibrated and equilibrated solvent modes were taken into theoretical consideration. All energetic calculations were performed at the M062x/6-31++G** level of theory, while for geometry optimized ONIOM methodology was used. The lowest adiabatic ionization potential was assigned for DNA containing a dA:dGOXO pair. Moreover, the adiabatic electron affinity was assigned at the same level for the mismatched and lesioned ds-oligo. Surprisingly, in the non-equilibrated mode, a significantly higher vertical electro affinity was found for lesioned DNA. The higher VEA in a non-equilibrated solvent state supported faster recognition in the A:GOXO base pair than A:G by MutY glycosylases under electron transfer mechanism.


Assuntos
DNA/química , Teoria da Densidade Funcional , Desoxiguanosina/química , Pareamento Incorreto de Bases , Dano ao DNA , Eletrônica
11.
Nucleic Acids Res ; 49(6): 3308-3321, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33677508

RESUMO

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the ß-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.


Assuntos
Caulobacter crescentus/genética , Reparo de Erro de Pareamento de DNA , Replicação do DNA , Motivos de Aminoácidos , Pareamento Incorreto de Bases , Caulobacter crescentus/metabolismo , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Fase S/genética
12.
Biochemistry ; 60(11): 873-885, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33689312

RESUMO

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions and mismatches in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Altered local dynamics and conformational properties in damaged DNAs have previously been suggested to assist in recognition and specificity. Herein, we use solution nuclear magnetic resonance to quantify changes in BI-BII backbone conformational dynamics due to the presence of single-base lesions in DNA, including uracil, dihydrouracil, 1,N6-ethenoadenine, and T:G mismatches. Stepwise changes to the %BII and ΔG of the BI-BII dynamic equilibrium compared to those of unmodified sequences were observed. Additionally, the equilibrium skews toward endothermicity for the phosphates nearest the lesion/mismatched base pair. Finally, the phosphates with the greatest alterations correlate with those most relevant to the repair of enzyme binding. All of these results suggest local conformational rearrangement of the DNA backbone may play a role in lesion recognition by repair enzymes.


Assuntos
Pareamento Incorreto de Bases , DNA/genética , DNA/metabolismo , Sítios de Ligação , DNA/química , DNA Glicosilases/metabolismo , Reparo do DNA , Humanos , Mutagênese , Conformação de Ácido Nucleico , Ligação Proteica
13.
J Phys Chem Lett ; 12(10): 2547-2554, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33683888

RESUMO

Monitoring the DNA dynamics in solution has great potential to develop new nucleic acid-based sensors and devices. With spectroscopic approaches, both at the ensemble average and single-molecule resolution, this study is directed to differentiate a single nucleotide mismatch (SNM) via a metal ion-stabilized mismatched base-pairing (C-Ag+-C/C-Cu2+-T) (C = cytosine, T = thymine) and site-selective extrinsic fluorophore, specifically, Thioflavin T (ThT). This is the first approach of its kind where dynamic quantities like molecular diffusion coefficients and diffusion times have been utilized to distinguish the least-stable SNM (CC & CT) formed by the most discriminating nucleobase, specifically, cytosine in a 20-mer duplex DNA. Additionally, this work also quantifies metal ions (Ag+ and Cu2+) at lower concentrations using fluorescence correlation spectroscopy. Our results can provide greater molecular-level insights into the mismatch-dependent metal-DNA interactions and also illuminate ThT as a new fluorophore to monitor the dynamics involved in DNA-metal composites.


Assuntos
Benzotiazóis/química , Cobre/química , DNA/química , Prata/química , Pareamento Incorreto de Bases , Pareamento de Bases , Íons/química , Espectrometria de Fluorescência
14.
J Microbiol ; 59(4): 401-409, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33779953

RESUMO

Eukaryotic genomes contain many duplicated genes closely located with each other, such as the hexose transporter (HXT) genes in Saccharomyces cerevisiae. They can potentially recombine via single-strand annealing (SSA) pathway. SSA between highly divergent sequences generates heteroduplex DNA intermediates with many mismatches, which can be corrected by mismatch repair (MMR), resulting in recombinant sequences with a single junction point. In this report, we demonstrate that SSA between HXT1 and HXT4 genes in MMR-deficient yeast cells produces recombinant genes with multiple-junctions resulting from alternating HXT1 and HXT4 tracts. The mutations in MMR genes had differential effects on SSA frequencies; msh6Δ mutation significantly stimulated SSA events, whereas msh2Δ and msh3Δ slightly suppressed it. We set up an assay that can identify a pair of recombinant genes derived from a single heteroduplex DNA. As a result, the recombinant genes with multiple-junctions were found to accompany genes with single-junctions. Based on the results presented here, a model was proposed to generate multiple-junctions in SSA pathway involving an alternative short-patch repair system.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Transporte de Monossacarídeos/genética , Ácidos Nucleicos Heteroduplexes/genética , Saccharomyces cerevisiae/genética , Pareamento Incorreto de Bases , DNA Fúngico , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mutação , Recombinação Genética
15.
Mol Cell Probes ; 56: 101707, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609730

RESUMO

BACKGROUND: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. METHODS: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 ∘C to the fully matched reference temperature. RESULTS: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. CONCLUSIONS: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.


Assuntos
Pareamento Incorreto de Bases , Primers do DNA/metabolismo , Sondas de DNA/metabolismo , DNA Viral/metabolismo , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , Genoma Viral , Alinhamento de Sequência , Temperatura
16.
Nucleic Acids Res ; 49(3): 1263-1277, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33503261

RESUMO

As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA/metabolismo , Pareamento Incorreto de Bases , Sistemas CRISPR-Cas , DNA/metabolismo , Escherichia coli/genética , Modelos Genéticos , Ligação Proteica , RNA/química , Termodinâmica
17.
Biopolymers ; 112(1): e23393, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32896905

RESUMO

T4 DNA ligase is a widely used ligase in many applications; yet in single nucleotide polymorphism analysis, it has been found generally lacking owing to its tendency to ligate mismatches quite efficiently. To address this lack of selectivity, we explored the effect of temperature on the selectivity of the ligase in discriminating single base pair mismatches at the 3'-terminus of the ligating strand using short ligation probes (9-mers). Remarkably, we observe outstanding selectivities when the assay temperature is increased to 7 °C to 13 °C above the dissociation temperature of the matched probe:target duplexes using commercially available enzyme at low concentration. Higher enzyme concentration shifts the temperature range to 13 °C to 19 °C above the probe:target dissociation temperatures. Finally, substituting the 5'-phosphate terminus with an abasic nucleotide decreases the optimal temperature range to 7 °C to 10 °C above the matched probe:target duplex. We compare the temperature dependence of the T4 DNA ligase catalyzed ligation and a nonenzymatic ligation system to contrast the origin of their modes of selectivity. For the latter, temperatures above the probe:target duplex dissociation lead to lower ligation conversions even for the perfect matched system. This difference between the two ligation systems reveals the uniqueness of the T4 DNA ligase's ability to maintain excellent ligation yields for the matched system at elevated temperatures. Although our observations are consistent with previous mechanistic work on T4 DNA ligase, by mapping out the temperature dependence for different ligase concentrations and probe modifications, we identify simple strategies for introducing greater selectivity into SNP discrimination based on ligation yields.


Assuntos
DNA Ligases/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Pareamento Incorreto de Bases , Reação de Cicloadição , Fluoresceína/química , Oligodesoxirribonucleotídeos/química , Temperatura de Transição
18.
Nat Commun ; 11(1): 5320, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087730

RESUMO

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Pareamento Incorreto de Bases , Proteínas de Ciclo Celular/metabolismo , Magnoliopsida/genética , Magnoliopsida/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Processamento Pós-Transcricional do RNA , RNA de Plantas/química , Ribonuclease III/metabolismo
19.
Future Microbiol ; 15: 1343-1352, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33085541

RESUMO

Aim: The inference of coronavirus evolution is largely based on mutations in SARS-CoV-2 genome. Misinterpretation of these mutations would mislead people about the evolution of SARS-CoV-2. Materials & methods: With 4521 lines of SARS-CoV-2, we obtained 3169 unique point mutation sites. We counted the numbers and calculated the minor allele frequency (MAF) of each mutation type. Results: Nearly half of the point mutations are C-T mismatches and 20% are A-G mismatches. The MAF of C-T and A-G mismatches is significantly higher than MAF of other mutation types. Conclusion: The excessive C-T mismatches do not resemble the random mutation profile. They are likely to be caused by the cytosine-to-uridine deamination system in hosts.


Assuntos
Betacoronavirus/genética , Mutação , RNA Viral/metabolismo , Pareamento Incorreto de Bases , COVID-19 , Uso do Códon , Infecções por Coronavirus/virologia , Citosina/metabolismo , Bases de Dados Genéticas , Desaminação , Frequência do Gene , Genoma Viral , Humanos , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , SARS-CoV-2 , Uridina/metabolismo
20.
Nucleic Acids Res ; 48(20): 11322-11334, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33080020

RESUMO

Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.


Assuntos
Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , DNA/química , Simulação de Dinâmica Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento de Bases , Biologia Computacional , DNA/metabolismo , Ligação de Hidrogênio , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Termodinâmica
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