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1.
Biochemistry ; 60(41): 3086-3097, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34613715

RESUMO

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.


Assuntos
Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Pressão Hidrostática , Ligação Proteica , Desdobramento de Proteína , Temperatura , Termodinâmica
2.
Nat Commun ; 12(1): 5966, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645815

RESUMO

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.


Assuntos
Proteína BRCA2/genética , DNA Primase/genética , DNA de Neoplasias/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Enzimas Multifuncionais/genética , Reparo de DNA por Recombinação , Proteína BRCA2/antagonistas & inibidores , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/antagonistas & inibidores , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas de Manutenção de Minicromossomo/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
J Phys Chem Lett ; 12(37): 9132-9141, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34523927

RESUMO

Different nucleotides generate specific ionic currents that discriminate between the nucleotides while they are passing through the nanopore constriction. MspA is a commonly used nanopore for DNA sequencing. However, the reasons of the current variation remain ambiguous. Our work unveils the microscopic mechanism of current variation for an ssDNA passing through the MspA nanopore by all-atom molecular dynamic simulations. Besides the physical rigidity and dimensions of the nucleotides, nucleotide orientation is observed to induce nonignorable current variation. Besides the generally considered MspA nanopore constriction, it is also found that the region below constriction could be used to detect and differentiate single nucleotides when the single-stranded DNA translocates in the form of base-constriction-base meshing and ratcheting across the nanopore constriction compared to other regions. The work provides a novel insight into facilitating the development of low-cost and high-throughput nanopore DNA sequencing.


Assuntos
Nanoporos , Porinas/química , Análise de Sequência de DNA/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/metabolismo , Porinas/metabolismo
4.
Nat Commun ; 12(1): 5545, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545070

RESUMO

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nucleotídeos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Homologia de Sequência de Aminoácidos , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , DNA de Cadeia Simples/metabolismo , Fluorescência , Interferometria , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Especificidade da Espécie
5.
Sci Rep ; 11(1): 17682, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480058

RESUMO

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA-protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein's hydration shell via soft interactions that, in turn, tune protein-ligand binding dynamics and affinity.


Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Espectroscopia de Ressonância Magnética , Ligação Proteica
6.
Nature ; 597(7876): 426-429, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471288

RESUMO

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Biológicos , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Homologia de Sequência do Ácido Nucleico , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Fatores de Tempo
7.
Molecules ; 26(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34576930

RESUMO

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/administração & dosagem , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/administração & dosagem , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , DNA de Cadeia Simples/metabolismo , Relação Dose-Resposta a Droga , Rad51 Recombinase/antagonistas & inibidores
8.
Biomolecules ; 11(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34439828

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.


Assuntos
Doenças Autoimunes/terapia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Metilação de DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Engenharia Genética/métodos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Guia/genética , RNA Guia/metabolismo
9.
Nucleic Acids Res ; 49(13): 7628-7643, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197611

RESUMO

Many type III CRISPR-Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.


Assuntos
Sistemas CRISPR-Cas , Desoxirribonucleases/metabolismo , Transcrição Genética , Trifosfato de Adenosina/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Clivagem do DNA , DNA de Cadeia Simples/metabolismo , Ligantes , Plasmídeos/metabolismo , RNA/análise
10.
Nucleic Acids Res ; 49(14): 7870-7883, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34283224

RESUMO

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.


Assuntos
Compostos Azo/metabolismo , Atrofia Muscular Espinal/genética , Pirimidinas/metabolismo , Precursores de RNA/genética , Splicing de RNA , Compostos Azo/química , Compostos Azo/uso terapêutico , Sequência de Bases , Sítios de Ligação/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Éxons/genética , Cinética , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Estrutura Molecular , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/metabolismo , Mutação , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/uso terapêutico , Conformação de Ácido Nucleico , Pirimidinas/química , Pirimidinas/uso terapêutico , Precursores de RNA/química , Precursores de RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética
11.
J Mol Biol ; 433(18): 167147, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34246654

RESUMO

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17-18 nucleotides and can melt at least 10-11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecBNuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecBNuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecBNuc. This suggests that the RecBNuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecBNuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecBNuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.


Assuntos
Pareamento de Bases , DNA de Cadeia Simples/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Exodesoxirribonuclease V/metabolismo , Desnaturação de Ácido Nucleico , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/genética , Estrutura Molecular , Conformação Proteica , Recombinação Genética , Termodinâmica
12.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
13.
Chem Commun (Camb) ; 57(52): 6416-6419, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34095911

RESUMO

Herein, based on iron oxyhydroxide (FeOOH) as the photoactive material and exonuclease III (exo III)-aided dual recycling signal amplification, a new photoelectrochemical (PEC) biosensor was successfully developed for human papillomavirus-16 (HPV-16) detection with a wide linear range from 0.5 fM to 1 nM and a low detection limit of 0.17 fM.


Assuntos
Técnicas Biossensoriais/métodos , Exodesoxirribonucleases/metabolismo , Compostos Férricos/química , Papillomavirus Humano 16/isolamento & purificação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Técnicas Eletroquímicas , Eletrodos , Papillomavirus Humano 16/genética , Humanos , Luz , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Reprodutibilidade dos Testes
14.
J Mol Biol ; 433(15): 167072, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34081984

RESUMO

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.


Assuntos
DNA Helicases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Microscopia de Fluorescência , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Imagem Individual de Molécula
15.
Elife ; 102021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34159897

RESUMO

Targeting DNA repair pathway offers an important therapeutic strategy for Homo sapiens (human) cancers. However, the failure of DNA repair inhibitors to markedly benefit patients necessitates the development of new strategies. Here, we show that exosome component 1 (EXOSC1) promotes DNA damages and sensitizes human kidney renal clear cell carcinoma (KIRC) cells to DNA repair inhibitor. Considering that endogenous source of mutation (ESM) constantly assaults genomic DNA and likely sensitizes human cancer cells to the inhibitor, we first analyzed the statistical relationship between the expression of individual genes and the mutations for KIRC. Among the candidates, EXOSC1 most notably promoted DNA damages and subsequent mutations via preferentially cleaving C site(s) in single-stranded DNA. Consistently, EXOSC1 was more significantly correlated with C>A transversions in coding strands than these in template strands in human KIRC. Notably, KIRC patients with high EXOSC1 showed a poor prognosis, and EXOSC1 sensitized human cancer cells to poly(ADP-ribose) polymerase inhibitors. These results show that EXOSC1 acts as an ESM in KIRC, and targeting EXOSC1 might be a potential therapeutic strategy.


Assuntos
Carcinoma de Células Renais/metabolismo , DNA de Cadeia Simples/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Neoplasias Renais/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , Camundongos , Camundongos Nus , Proteínas de Ligação a RNA/metabolismo
16.
Biochem Biophys Res Commun ; 567: 190-194, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34166917

RESUMO

Pif1 helicases, conserved in eukaryotes, are involved in maintaining genome stability in both the nucleus and mitochondria. Here, we report the crystal structure of a truncated Candida Albicans Pif1 (CaPif1368-883) in complex with ssDNA and an ATP analog. Our results show that the Q-motif is responsible for identifying adenine bases, and CaPif1 preferentially utilizes ATP/dATP during dsDNA unwinding. Although CaPif1 shares structural similarities with Saccharomyces cerevisiae Pif1, CaPif1 can contact the thymidine bases of DNA by hydrogen bonds, whereas ScPif1 cannot. More importantly, the crosslinking and mutant experiments have demonstrated that the conformational change of domain 2B is necessary for CaPif1 to unwind dsDNA. These findings contribute to further the understanding of the unwinding mechanism of Pif1.


Assuntos
Candida albicans/metabolismo , DNA Helicases/metabolismo , Proteínas Fúngicas/metabolismo , Trifosfato de Adenosina/metabolismo , Candida albicans/química , Candidíase/microbiologia , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , DNA de Cadeia Simples/metabolismo , Proteínas Fúngicas/química , Humanos , Modelos Moleculares , Conformação Proteica
17.
J Virol ; 95(17): e0046721, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34106772

RESUMO

We previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense single-stranded RNA (ssRNA) virus, yadokari virus 1 (YkV1), and an unrelated double-stranded RNA (dsRNA) virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix. We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp and whether YnV1 capsid copackages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs in the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein cotranslationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsids solely packaging YkV1 dsRNA and RdRp. These results provide proof of concept that a capsidless positive-sense ssRNA [(+)ssRNA] virus is hosted by an unrelated dsRNA virus. IMPORTANCE Viruses typically encode their own capsids that encase their genomes. However, a capsidless positive-sense single-stranded RNA [(+)ssRNA] virus, YkV1, depends on an unrelated double-stranded RNA (dsRNA) virus, YnV1, for encapsidation and replication. We previously showed that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the heterocapsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains of the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsids solely packaging YkV1 dsRNA and RdRp. This study provides proof of concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.


Assuntos
Ascomicetos/virologia , Capsídeo/metabolismo , DNA de Cadeia Simples/metabolismo , Micovírus/enzimologia , Vírus de RNA/enzimologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , DNA de Cadeia Simples/genética , Micovírus/genética , Genoma Viral , Mutação , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação , Montagem de Vírus , Replicação Viral
18.
Chem Commun (Camb) ; 57(48): 5941-5944, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34018521

RESUMO

This work describes a single-stranded degradable modular grafting probe for analyzing microRNA-21. In the system, the exonuclease activity of phi29 polymerase restrains the SYBR Green I/ssDNA induced background. The palindrome activation caused remarkable target fluorescence. The detection limit was achieved as 0.26 fM, showing potential in biochemical analysis.


Assuntos
Benzotiazóis/química , DNA de Cadeia Simples/química , Diaminas/química , Exodesoxirribonucleases/química , MicroRNAs/análise , Quinolinas/química , Benzotiazóis/metabolismo , DNA de Cadeia Simples/metabolismo , Diaminas/metabolismo , Exodesoxirribonucleases/metabolismo , Fluorescência , Humanos , MicroRNAs/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Quinolinas/metabolismo
19.
Bioconjug Chem ; 32(6): 1156-1166, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34009954

RESUMO

Fluorescent antibodies are a workhorse of biomedical science, but fluorescence multiplexing has been notoriously difficult due to spectral overlap between fluorophores. We recently established proof-of-principal for fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which uses combinations of existing fluorophores to create unique spectral signatures for increased multiplexing. However, a method for labeling antibodies with MuSIC probes has not yet been developed. Here, we present a method for labeling antibodies with MuSIC probes. We conjugate a DBCO-Peg5-NHS ester linker to antibodies and a single-stranded DNA "docking strand" to the linker and, finally, hybridize two MuSIC-compatible, fluorescently labeled oligos to the docking strand. We validate the labeling protocol with spin-column purification and absorbance measurements. We demonstrate the approach using (i) Cy3, (ii) Tex615, and (iii) a Cy3-Tex615 combination as three different MuSIC probes attached to three separate batches of antibodies. We created single-, double-, and triple-positive beads that are analogous to single cells by incubating MuSIC probe-labeled antibodies with protein A beads. Spectral flow cytometry experiments demonstrate that each MuSIC probe can be uniquely distinguished, and the fraction of beads in a mixture with different staining patterns are accurately inferred. The approach is general and might be more broadly applied to cell-type profiling or tissue heterogeneity studies in clinical, biomedical, and drug discovery research.


Assuntos
Anticorpos/química , Anticorpos/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Espectrometria de Fluorescência
20.
DNA Repair (Amst) ; 104: 103132, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34049076

RESUMO

Lack of coordination between the DNA replication and transcription machineries can increase the frequency of transcription-replication conflicts, leading ultimately to DNA damage and genomic instability. A major source of these conflicts is the formation of R-loops, which consist of a transcriptionally generated RNA-DNA hybrid and the displaced single-stranded DNA. R-loops play important physiological roles and have been implicated in human diseases. Although these structures have been extensively studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. We found that in cancer cells, tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) interacted with PARP1 and localized to R-loops in response to DNA-damaging agent camptothecin (CPT), which is associated with R-loop formation. PARP1-mediated PARylation was required for recruitment of TonEBP to the sites of R-loop-associated DNA damage. Loss of TonEBP increased levels of R-loop accumulation and DNA damage, and promoted cell death in response to CPT. These findings suggest that TonEBP mediates resistance to CPT-induced cell death by preventing R-loop accumulation in cancer cells.


Assuntos
Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Estruturas R-Loop , Fatores de Transcrição/metabolismo , Transcrição Genética , Camptotecina/toxicidade , Linhagem Celular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Poli ADP Ribosilação
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