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1.
Cell ; 174(5): 1188-1199.e14, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30057118

RESUMO

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.


Assuntos
DNA Bacteriano , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/metabolismo , Microscopia de Fluorescência , Poliestirenos/química , Proteoma , Análise de Sequência de RNA , Estresse Mecânico , Transcriptoma
2.
Mol Cell ; 81(15): 3033-3037, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358454

RESUMO

Some biological questions are tough to solve through standard molecular and cell biological methods and naturally lend themselves to investigation by physical approaches. Below, a group of formally trained physicists discuss, among other things, how they apply physics to address biological questions and how physical approaches complement conventional biological approaches.


Assuntos
Biofísica/métodos , Modelos Biológicos , Física/métodos , Imagem Individual de Molécula , Biologia/educação , Biofísica/tendências , Cromossomos/química , Cromossomos/ultraestrutura , Simulação por Computador , Humanos , Proteínas Motores Moleculares/química , Origem da Vida , Física/educação , Imagem Individual de Molécula/métodos
3.
Mol Cell ; 81(21): 4467-4480.e7, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34687604

RESUMO

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.


Assuntos
Pirazinas/química , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Recombinação Genética , Ribonucleotídeos/química , Animais , Antivirais , Catálise , Células Cultivadas , Técnicas Genéticas , Genoma , Genoma Viral , Recombinação Homóloga , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Mutagênese , Nucleotídeos/genética , Conformação Proteica , RNA/química , RNA Polimerase Dependente de RNA/metabolismo , RNA-Seq , Transgenes , Virulência
4.
Cell ; 142(4): 519-30, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20723754

RESUMO

Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/química , Animais , Fenômenos Fisiológicos Celulares , DNA/química , DNA/metabolismo , DNA Super-Helicoidal/metabolismo , Humanos
5.
Nucleic Acids Res ; 51(8): 3770-3792, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36942484

RESUMO

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.


Assuntos
Cromatina , Histonas , Histonas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Cromatina/genética , Replicação do DNA , DNA/genética
6.
Biophys J ; 123(1): 31-41, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-37968907

RESUMO

DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3' single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3' single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription.


Assuntos
Proteínas de Ciclo Celular , DNA de Cadeia Simples , Proteínas de Ciclo Celular/metabolismo , DNA/química , Replicação do DNA , Plasmídeos/genética
7.
Crit Rev Biochem Mol Biol ; 53(1): 49-63, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29108427

RESUMO

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus-Ter interactions at the nonpermissive face - interactions that result in a highly stable "locked" complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus-Ter mechanism - leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA-protein system further.


Assuntos
Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Bactérias/química , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mapas de Interação de Proteínas
8.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487277

RESUMO

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.


Assuntos
Enterovirus Humano A/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Surtos de Doenças , Enterovirus/genética , Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Genoma Viral/genética , Humanos , Mutação , Filogenia , Poliomielite/epidemiologia , Poliomielite/virologia , Estudos Retrospectivos , Virulência
9.
Biophys J ; 117(11): 2217-2227, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31521330

RESUMO

Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)2 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque buildup in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behavior. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.


Assuntos
Cromossomos/genética , DNA/genética , Animais , Sequência de Bases , Cromossomos/efeitos dos fármacos , DNA/química , Drosophila/genética , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Sais/farmacologia , Termodinâmica
10.
Nat Rev Genet ; 14(1): 9-22, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23150038

RESUMO

To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.


Assuntos
Genômica/métodos , Replicação do DNA , Fenômenos Genéticos , Genômica/tendências , Biossíntese de Proteínas , Transcrição Gênica
11.
Nucleic Acids Res ; 45(10): 5920-5929, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28460037

RESUMO

The mechanical properties of DNA fundamentally constrain and enable the storage and transmission of genetic information and its use in DNA nanotechnology. Many properties of DNA depend on the ionic environment due to its highly charged backbone. In particular, both theoretical analyses and direct single-molecule experiments have shown its bending stiffness to depend on salt concentration. In contrast, the salt-dependence of the twist stiffness of DNA is much less explored. Here, we employ optimized multiplexed magnetic torque tweezers to study the torsional stiffness of DNA under varying salt conditions as a function of stretching force. At low forces (<3 pN), the effective torsional stiffness is ∼10% smaller for high salt conditions (500 mM NaCl or 10 mM MgCl2) compared to lower salt concentrations (20 mM NaCl and 100 mM NaCl). These differences, however, can be accounted for by taking into account the known salt dependence of the bending stiffness. In addition, the measured high-force (6.5 pN) torsional stiffness values of C = 103 ± 4 nm are identical, within experimental errors, for all tested salt concentration, suggesting that the intrinsic torsional stiffness of DNA does not depend on salt.


Assuntos
DNA/química , Cloreto de Magnésio/química , Nanotecnologia/instrumentação , Imagem Individual de Molécula/métodos , Cloreto de Sódio/química , Fenômenos Biomecânicos , Dureza , Cinética , Campos Magnéticos , Nanotecnologia/métodos , Conformação de Ácido Nucleico , Pinças Ópticas , Imagem Individual de Molécula/instrumentação , Eletricidade Estática , Termodinâmica , Torção Mecânica
12.
Proc Natl Acad Sci U S A ; 113(18): 4982-7, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27091987

RESUMO

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA-binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Modelos Teóricos , Concentração de Íons de Hidrogênio , Magnésio/química , Sais/química
13.
Biophys J ; 114(8): 1970-1979, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29694873

RESUMO

Single-molecule manipulation techniques have provided unprecedented insights into the structure, function, interactions, and mechanical properties of biological macromolecules. Recently, the single-molecule toolbox has been expanded by techniques that enable measurements of rotation and torque, such as the optical torque wrench (OTW) and several different implementations of magnetic (torque) tweezers. Although systematic analyses of the position and force precision of single-molecule techniques have attracted considerable attention, their angle and torque precision have been treated in much less detail. Here, we propose Allan deviation as a tool to systematically quantitate angle and torque precision in single-molecule measurements. We apply the Allan variance method to experimental data from our implementations of (electro)magnetic torque tweezers and an OTW and find that both approaches can achieve a torque precision better than 1 pN · nm. The OTW, capable of measuring torque on (sub)millisecond timescales, provides the best torque precision for measurement times ≲10 s, after which drift becomes a limiting factor. For longer measurement times, magnetic torque tweezers with their superior stability provide the best torque precision. Use of the Allan deviation enables critical assessments of the torque precision as a function of measurement time across different measurement modalities and provides a tool to optimize measurement protocols for a given instrument and application.


Assuntos
Estatística como Assunto/métodos , Torque , Fenômenos Magnéticos
14.
J Biol Chem ; 292(42): 17506-17513, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28855255

RESUMO

The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.


Assuntos
DNA/química , Histonas/química , Nucleossomos/química , Dobramento de Proteína , Proteínas de Xenopus/química , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Proteínas de Xenopus/metabolismo , Xenopus laevis
16.
J Chem Phys ; 148(12): 123323, 2018 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-29604863

RESUMO

Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.


Assuntos
Histonas/química , Histonas/classificação , Iodoacetamida/química , Conformação Molecular
17.
Nucleic Acids Res ; 44(13): 6262-73, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27166373

RESUMO

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.


Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Segregação de Cromossomos/genética , Cromossomos Bacterianos/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Substâncias Macromoleculares/metabolismo
18.
Nat Chem Biol ; 11(8): 579-85, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26147356

RESUMO

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.


Assuntos
Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/química , DNA Circular/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
19.
Methods ; 105: 90-8, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27038745

RESUMO

Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20pN) for prolonged periods of time (>1000s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.


Assuntos
DNA Helicases/química , Proteínas de Ligação a DNA/química , Ensaios de Triagem em Larga Escala/métodos , Imagem Individual de Molécula/métodos , DNA/química , DNA Helicases/isolamento & purificação , Proteínas de Ligação a DNA/genética , Pinças Ópticas , Vírus 40 dos Símios/enzimologia
20.
Nucleic Acids Res ; 43(21): 10421-9, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26496948

RESUMO

Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3'-end that allows for the de novo initiation of a second RdRp. This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.


Assuntos
Bacteriófago phi 6/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Sítio de Iniciação de Transcrição , Moldes Genéticos , Transcrição Gênica
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