RESUMEN
The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.
Asunto(s)
Proteínas Portadoras , ADN , Proteínas Nucleares , Unión Proteica , Humanos , ADN/metabolismo , ADN/genética , ADN/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Mutación , Dominios Proteicos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , Roturas del ADN de Doble Cadena , Sitios de UniónRESUMEN
Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.
Asunto(s)
Betacoronavirus , ARN Viral , Betacoronavirus/genética , Microscopía por Crioelectrón , Genoma Viral/genética , ARN Viral/química , ARN Viral/genética , ARN Viral/ultraestructura , SARS-CoV-2/genéticaRESUMEN
Genome integrity and maintenance are essential for the viability of all organisms. A wide variety of DNA damage types have been described, but double-strand breaks (DSBs) stand out as one of the most toxic DNA lesions. Two major pathways account for the repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Both pathways involve complex DNA transactions catalyzed by proteins that sequentially or cooperatively work to repair the damage. Single-molecule methods allow visualization of these complex transactions and characterization of the protein:DNA intermediates of DNA repair, ultimately allowing a comprehensive breakdown of the mechanisms underlying each pathway. We review current understanding of the HR and NHEJ responses to DSBs in eukaryotic cells, with a particular emphasis on recent advances through the use of single-molecule techniques.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Reparación del ADN/genética , ADN/genética , Daño del ADN , Reparación del ADN por Unión de Extremidades/genéticaRESUMEN
The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.
Asunto(s)
Candida glabrata , Proteínas de Unión a Telómeros , Humanos , Candida glabrata/genética , Candida glabrata/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Unión Proteica , Complejo Shelterina , Telómero/genética , Telómero/metabolismoRESUMEN
The synapsis of DNA ends is a critical step for the repair of double-strand breaks by non-homologous end joining (NHEJ). This is performed by a multicomponent protein complex assembled around Ku70-Ku80 heterodimers and regulated by accessory factors, including long non-coding RNAs, through poorly understood mechanisms. Here, we use magnetic tweezers to investigate the contributions of core NHEJ proteins and APLF and lncRNA NIHCOLE to DNA synapsis. APLF stabilizes DNA end bridging and, together with Ku70-Ku80, establishes a minimal complex that supports DNA synapsis for several minutes under piconewton forces. We find the C-terminal acidic region of APLF to be critical for bridging. NIHCOLE increases the dwell time of the synapses by Ku70-Ku80 and APLF. This effect is further enhanced by a small and structured RNA domain within NIHCOLE. We propose a model where Ku70-Ku80 can simultaneously bind DNA, APLF, and structured RNAs to promote the stable joining of DNA ends.
Asunto(s)
ARN Largo no Codificante , ARN Largo no Codificante/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Roturas del ADN de Doble Cadena , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Reparación del ADN por Unión de Extremidades , ADN/metabolismo , Reparación del ADNRESUMEN
Single molecule biophysics experiments for the study of DNA-protein interactions usually require production of a homogeneous population of long DNA molecules with controlled sequence content and/or internal tertiary structures. Traditionally, Lambda phage DNA has been used for this purpose, but it is difficult to customize. In this article, we provide a detailed and simple protocol for cloning large (~25kbp) plasmids with bespoke sequence content, which can be used to generate custom DNA constructs for a range of single-molecule experiments. In particular, we focus on a procedure for making long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids and long DNA constructs with flaps, which are especially relevant for studying the activity of DNA helicases and translocases. Additionally, we describe how the modification of the free ends of such substrates can facilitate their binding to functionalized surfaces allowing immobilization and imaging using dual optical tweezers and confocal microscopy. Finally, we provide examples of how these DNA constructs have been applied to study the activity of human DNA helicase B (HELB). The techniques described herein are simple, versatile, adaptable, and accessible to any laboratory with access to standard molecular biology methods.
Asunto(s)
Ácidos Nucleicos , Pinzas Ópticas , ADN/química , ADN Helicasas/metabolismo , ADN de Cadena Simple , HumanosRESUMEN
Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPAsingle-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of â¼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5' to 3' direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.
Asunto(s)
ADN Helicasas , ADN de Cadena Simple , Proteínas Motoras Moleculares , Proteína de Replicación A , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Humanos , Hidrólisis , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Unión Proteica , Proteína de Replicación A/metabolismoRESUMEN
Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citidina Trifosfato/metabolismo , ADN Bacteriano/metabolismo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Centrómero/metabolismo , Segregación Cromosómica , Cromosomas Bacterianos , Hidrólisis , Unión Proteica , Pirofosfatasas/metabolismoRESUMEN
DNA dynamics can only be understood by taking into account its complex mechanical behavior at different length scales. At the micrometer level, the mechanical properties of single DNA molecules have been well-characterized by polymer models and are commonly quantified by a persistence length of 50 nm (~150 bp). However, at the base pair level (~3.4 Å), the dynamics of DNA involves complex molecular mechanisms that are still being deciphered. Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective. We first discuss recent findings on sequence-dependent DNA mechanical properties, including sequences that resist mechanical stress and sequences that can accommodate strong deformations. We then comment on the intricate effects of cytosine methylation and DNA mismatches on DNA mechanics. Finally, we review recently reported differences in the mechanical properties of DNA and double-stranded RNA, the other double-helical carrier of genetic information. A thorough examination of the recent single-molecule literature permits establishing a set of general 'rules' that reasonably explain the mechanics of nucleic acids at the base pair level. These simple rules offer an improved description of certain biological systems and might serve as valuable guidelines for future design of DNA and RNA nanostructures.
Asunto(s)
ADN , Nanotecnología , Emparejamiento Base , Conformación de Ácido Nucleico , ARN BicatenarioRESUMEN
Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. SIGNIFICANCE: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.See related commentary by Barcena-Varela and Lujambio, p. 4899.
Asunto(s)
Carcinoma Hepatocelular/genética , Roturas del ADN de Doble Cadena , Neoplasias Hepáticas/genética , ARN Largo no Codificante/genética , Biomarcadores de Tumor , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidad , Modelos Biológicos , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Pronóstico , ARN Largo no Codificante/químicaRESUMEN
Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
Asunto(s)
Proteínas de Ciclo Celular/genética , Complejos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Fenómenos Biofísicos , Proteínas de Ciclo Celular/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/ultraestructura , Proteínas de Unión al ADN/genética , Humanos , Interfase/genética , Mitosis/genética , Complejos Multiproteicos/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Sumoilación/genética , CohesinasRESUMEN
Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix. Using all-atom molecular dynamics simulations, we identified a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that strongly bend the RNA double-helix. This finding was experimentally validated using atomic force microscopy imaging of dsRNA molecules designed to display macroscopic curvature via repetitions of phased AU-tract motifs. At the atomic level, this novel phenomenon originates from a localized compression of the dsRNA major groove and a large propeller twist at the position of the AU-tract. Moreover, the magnitude of the bending can be modulated by changing the length of the AU-tract. Altogether, our results demonstrate the possibility of modifying the dsRNA curvature by means of its nucleotide sequence, which may be exploited in the emerging field of RNA nanotechnology and might also constitute a natural mechanism for proteins to achieve recognition of specific dsRNA sequences.
Asunto(s)
Adenina/química , ADN/genética , ARN Bicatenario/genética , Uracilo/química , ADN/química , ADN/ultraestructura , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , ARN Bicatenario/química , ARN Bicatenario/ultraestructuraRESUMEN
Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level.
RESUMEN
DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of â¼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Geobacillus stearothermophilus/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , ADN , ADN Helicasas/química , ADN Helicasas/aislamiento & purificación , Desoxirribonucleasa I/química , Desoxirribonucleasa I/aislamiento & purificaciónRESUMEN
Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.
Asunto(s)
Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Líquido Amniótico/metabolismo , Asparagina/metabolismo , Unión Competitiva , Cromatografía de Afinidad , Femenino , Glicosilación , Humanos , Polisacáridos/metabolismo , Embarazo , Unión Proteica , Multimerización de Proteína , Proteinosis Alveolar Pulmonar/metabolismo , Proteína D Asociada a Surfactante Pulmonar/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, "fuzzy balls". However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50-60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein.
Asunto(s)
Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Animales , Células CHO , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Cricetulus , Escherichia coli/química , Expresión Génica , Humanos , Agregado de Proteínas , Unión Proteica , Multimerización de Proteína , Proteína D Asociada a Surfactante Pulmonar/biosíntesis , Proteínas Recombinantes/biosíntesisRESUMEN
A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (â¼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.
Asunto(s)
Secuencia Rica en At , ADN de Helmintos/química , Animales , Fenómenos Biomecánicos , Caenorhabditis elegans/genética , ADN de Helmintos/ultraestructura , Genoma de los Helmintos , Microscopía de Fuerza Atómica , Pinzas ÓpticasRESUMEN
The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.
Asunto(s)
Proteínas Bacterianas/genética , ADN Helicasas/genética , Replicación del ADN/genética , Farmacorresistencia Bacteriana/genética , Roturas del ADN de Cadena Simple/efectos de los fármacos , Proteínas de Unión al ADN/genética , Geobacillus stearothermophilus/efectos de los fármacos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/patogenicidad , Plásmidos/efectos de los fármacos , Plásmidos/genética , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Tetraciclina/farmacología , Transactivadores/genéticaRESUMEN
The mechanical properties of double-stranded RNA (dsRNA) are involved in many of its biological functions and are relevant for future nanotechnology applications. DsRNA must tightly bend to fit inside viral capsids or deform upon the interaction with proteins that regulate gene silencing or the immune response against viral attacks. However, the question of how the nucleotide sequence affects the global mechanical properties of dsRNA has so far remained largely unexplored. Here, we have employed state-of-the-art atomistic molecular dynamics simulations to unveil the mechanical response of different RNA duplexes to an external force. Our results reveal that, similarly to dsDNA, the mechanical properties of dsRNA are highly sequence-dependent. However, we find that the nucleotide sequence affects in a strikingly different manner the stretching and twisting response of RNA and DNA duplexes under force. We find that the elastic response of dsRNA is dominated by the local high flexibility of pyrimidine-purine steps. Moreover, the flexibility of pyrimidine-purine steps is independent of the sequence context, and the global flexibility of the duplex reasonably scales with the number of this kind of base-pair dinucleotides. We conclude that disparities of the mechanical response of dinucleotides are responsible for the differences observed in the mechanical properties of RNA and DNA duplexes.
Asunto(s)
Emparejamiento Base , Secuencia de Bases , Simulación de Dinámica Molecular , ARN Bicatenario/químicaRESUMEN
Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. Previously, we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualise ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerisation with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.