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1.
Enzymes ; 45: 183-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627877

RESUMO

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Complexos Multienzimáticos/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/química
2.
Enzymes ; 45: 59-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627883

RESUMO

The nucleotide excision repair (NER) system removes a variety of types of helix-distorting lesions from DNA through a dual incision mechanism, in which the damaged nucleotide bases are excised in the form of a small, excised, damage-containing single-stranded DNA oligonucleotide (sedDNA). Damage removal leaves a gap in the DNA template that must then be filled in by the action of a DNA polymerase and ligated to the downstream phosphodiester backbone in the DNA to complete the repair reaction. Defects in damage removal, sedDNA processing, or gap filling have the potential to be mutagenic and lethal to cells, and thus several human pathologies, including cancer and aging, are associated with defects in NER. This review summarizes our current understanding of NER with a focus on the enzymes that excise sedDNAs and restore the duplex DNA to its native state in human cells.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/química , DNA/metabolismo , DNA/biossíntese , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos
3.
Nat Commun ; 10(1): 4423, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562312

RESUMO

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Domínio Catalítico , Quebras de DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida
4.
Nat Commun ; 10(1): 4390, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558728

RESUMO

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Proteínas Argonauta/química , Proteínas Argonauta/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Clostridium butyricum/genética , Clostridium butyricum/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Difusão , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Cinética , Microscopia de Fluorescência/métodos , Ligação Proteica , Estrutura Secundária de Proteína , RNA/química , RNA/genética , Imagem Individual de Molécula/métodos
5.
J Dairy Sci ; 102(11): 9702-9710, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31477297

RESUMO

Monitoring Staphylococcus aureus with high sensitivity is very important for ensuring milk quality and food safety. In this study, we used a rapid nucleic acid isothermal amplification method, saltatory rolling circle amplification (SRCA), for the detection of Staph. aureus in milk. The results of the SRCA method can be assessed visually by the presence of white precipitate or by fluorescence measurement. Thirteen Staph. aureus strains and 31 non-Staph. aureus strains were used to evaluate the specificity of SRCA. The method exhibited excellent detection of Staph. aureus genomic DNA at a concentration of 7.8 × 101 fg/µL when assessed by visible precipitate, and at 7.8 × 100 fg/µL when detected by fluorescence after addition of the fluorochrome SYBR Green I. In artificially inoculated milk, the detection limits of SRCA were 5.6 × 102 cfu/mL by precipitate and 5.6 × 101 cfu/mL by fluorescence, respectively. Compared with conventional PCR approaches, the SRCA assay achieved at least 100-fold higher sensitivity. Moreover, the sensitivity, specificity, and accuracy of the SRCA-based system were calculated to be 100.00, 97.73, and 97.78%, respectively. These results indicate that SRCA has potential application as a sensitive and visual technique for the detection of Staph. aureus in milk.


Assuntos
Mastite Bovina/diagnóstico , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/isolamento & purificação , Animais , Bovinos , DNA/metabolismo , Primers do DNA/química , DNA Complementar/química , DNA de Cadeia Simples/metabolismo , Limite de Detecção , Mastite Bovina/microbiologia , Técnicas de Amplificação de Ácido Nucleico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética
6.
Nat Commun ; 10(1): 4058, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492866

RESUMO

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/química , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica , Mutação , Ligação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
7.
Nucleic Acids Res ; 47(15): 7798-7808, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31372639

RESUMO

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.


Assuntos
Trifosfato de Adenosina/química , DNA de Cadeia Simples/química , DNA/química , Recombinação Homóloga , Ácidos Nucleicos Heteroduplexes/química , Recombinases Rec A/química , Trifosfato de Adenosina/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sítios de Ligação , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Hidrólise , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Ligação Proteica , Conformação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo
8.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
9.
Nat Commun ; 10(1): 3854, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451692

RESUMO

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.


Assuntos
Exossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/metabolismo , Líquidos Corporais/citologia , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoconjugados/genética , Imunoconjugados/metabolismo , Proteínas de Membrana/genética , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Análise de Sequência de DNA/métodos
10.
PLoS Genet ; 15(8): e1008294, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31369547

RESUMO

The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
11.
Nucleic Acids Res ; 47(16): 8595-8605, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31340040

RESUMO

G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5'-3'-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3'-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.


Assuntos
DNA Helicases/genética , DNA Polimerase III/genética , Polimerase do DNA Mitocondrial/genética , DNA Fúngico/genética , Quadruplex G , Proteína de Replicação A/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , Polimerase do DNA Mitocondrial/metabolismo , Replicação do DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Genoma Fúngico , Instabilidade Genômica , Mitocôndrias/metabolismo , Ligação Proteica , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Nanoscale ; 11(30): 14426-14433, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31334729

RESUMO

Nanopore DNA sequencing with a solid-state nanopore requires deceleration of the ultrafast translocation speed of single-stranded DNA (ssDNA). We report an unexpected phenomenon: controlled dielectric breakdown (CBD) with a divalent metal cation, especially Ca2+, provides a silicon nitride nanopore with the ability to decelerate ssDNA speed to 100 µs per base even after solution replacement. This speed is two orders of magnitude slower than that for CBD with a conventional monovalent metal cation. Temperature dependence experiments revealed that the enthalpic barrier for a nanopore created via CBD with Ca2+ is 25-30kBT, comparable to that of a biological nanopore. The slowing effect originates from the strong interaction between ssDNA and divalent cations, which were coated on the sidewall of the nanopore during the CBD process. In addition, we found that the nanopore created via CBD with Ca2+ can decelerate the speed of even single-nucleotide monomers, dNMPs, to 0.1-10 ms per base. The four single nucleotides could be statistically identified according to their blockade currents. Our approach is simple and practical because it simultaneously allows nanopore fabrication, ssDNA deceleration and the identification of nucleotide monomers.


Assuntos
Nanoporos , Compostos de Silício/química , Cátions Bivalentes/química , Césio/química , Cloretos/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Nucleotídeos/análise , Análise de Sequência de DNA/métodos , Temperatura Ambiente
13.
Nucleic Acids Res ; 47(14): 7276-7293, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31318975

RESUMO

Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.


Assuntos
DNA de Cadeia Simples/química , DNA/química , Quadruplex G , Guanina/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Guanina/metabolismo , Humanos , Cinética
14.
Nat Commun ; 10(1): 3001, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278272

RESUMO

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Staphylococcus epidermidis/genética , Thermus thermophilus/genética , Elongação da Transcrição Genética/imunologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/imunologia , RNA Guia/genética , RNA Guia/imunologia , RNA Guia/metabolismo , Staphylococcus epidermidis/imunologia , Thermus thermophilus/imunologia
15.
Nucleic Acids Res ; 47(16): 8581-8594, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329947

RESUMO

Escherichia coli single strand (ss) DNA binding (SSB) protein protects ssDNA intermediates and recruits at least 17 SSB interacting proteins (SIPs) during genome maintenance. The SSB C-termini contain a 9 residue acidic tip and a 56 residue intrinsically disordered linker (IDL). The acidic tip interacts with SIPs; however a recent proposal suggests that the IDL may also interact with SIPs. Here we examine the binding to four SIPs (RecO, PriC, PriA and χ subunit of DNA polymerase III) of three peptides containing the acidic tip and varying amounts of the IDL. Independent of IDL length, we find no differences in peptide binding to each individual SIP indicating that binding is due solely to the acidic tip. However, the tip shows specificity, with affinity decreasing in the order: RecO > PriA ∼ χ > PriC. Yet, RecO binding to the SSB tetramer and an SSB-ssDNA complex show significant thermodynamic differences compared to the peptides alone, suggesting that RecO interacts with another region of SSB, although not the IDL. SSB containing varying IDL deletions show different binding behavior, with the larger linker deletions inhibiting RecO binding, likely due to increased competition between the acidic tip interacting with DNA binding sites within SSB.


Assuntos
DNA Helicases/química , DNA Polimerase III/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Genoma Bacteriano , Proteínas Intrinsicamente Desordenadas/química , Sequência de Aminoácidos , Sítios de Ligação , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
16.
Talanta ; 204: 424-430, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357315

RESUMO

It is very meaningful and useful to select specific aptamers with capacity to distinguish small structural analogues, but it is difficult to carry out by traditional affinity chromatography-SELEX (systematic evolution of ligands by exponential enrichment) based on immobilized target molecules. In this paper, as a proof of concept, we selected DNA aptamers that can specifically recognize and differentiate riboflavin and its derivative flavin adenine dinucleotide (FAD) by a modified method. Here, the random DNA library was indirectly immobilized on streptavidin functional agarose beads by hybridization with its biotinylated short complementary strand, and the specific affinity between aptamers and its target would induce the aptamers to release from beads. Binding specificity can be tailored by performing an additional negative SELEX with the structure analogue of target. After about 10 rounds of selection, 6 aptamers for riboflavin and 2 aptamers for FAD with good affinities were isolated, and their dissociation constants (Kds) were all at low micromolar level. Moreover, as expected, most of these aptamers show high affinity and excellent selectivity for target molecules, almost no binding to structure analogues and purines, indicating this simple method could be used to select specific aptamers to distinguish small molecular targets with similar structures.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA de Cadeia Simples/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Riboflavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Fluorescência , Estudo de Prova de Conceito , Riboflavina/química , Técnica de Seleção de Aptâmeros/métodos
17.
Nucleic Acids Res ; 47(14): 7494-7501, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31216020

RESUMO

Saccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 µM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a 'mobile' manner.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Modelos Biológicos , Conformação de Ácido Nucleico , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Mecânico
18.
Nucleic Acids Res ; 47(11): 5658-5669, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216043

RESUMO

ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.


Assuntos
ADP-Ribosilação , Difosfato de Adenosina/química , RNA/metabolismo , ADP Ribose Transferases/genética , Adenosina Difosfato Ribose , Animais , Catálise , Cromatina/química , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Camundongos , NAD/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Plasmídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
19.
Mol Cell ; 75(1): 145-153.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31153714

RESUMO

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.


Assuntos
DNA de Cadeia Simples/genética , Retroalimentação Fisiológica , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteína de Replicação A/genética , Sítios de Ligação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Humanos , Microscopia de Fluorescência , Nucleossomos/química , Nucleossomos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Ligação Proteica , RecQ Helicases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
20.
Nucleic Acids Res ; 47(14): 7321-7332, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31214713

RESUMO

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.


Assuntos
DNA de Cadeia Simples/genética , Inativação Gênica , MicroRNAs/genética , Ácidos Nucleicos Heteroduplexes/genética , Oligonucleotídeos Antissenso/genética , Animais , Northern Blotting , DNA de Cadeia Simples/metabolismo , Feminino , Regulação da Expressão Gênica , Rim/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos ICR , MicroRNAs/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/farmacocinética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/metabolismo
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