RESUMO
SMC (structural maintenance of chromosomes) protein complexes are an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. Here, we describe the roles of SMCs in chromosome biology and particularly review in vitro single-molecule studies that have recently advanced our understanding of SMC proteins. We describe the mechanistic biophysical aspects of loop extrusion that govern genome organization and its consequences.
Assuntos
Proteínas Cromossômicas não Histona , Complexos Multiproteicos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
With the focus on technology for this issue of Molecular Cell, a group of scientists working in different areas of molecular biology provide their perspective on the most recent important technological advance in their field, where the field is lacking, and their wish list for future technology development.
Assuntos
Pesquisa Biomédica/tendências , Técnicas Genéticas/tendências , Biologia Molecular/tendências , Animais , Difusão de Inovações , HumanosRESUMO
Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.
Assuntos
Centrômero , Proteínas Cromossômicas não Histona , Autoantígenos/genética , Autoantígenos/metabolismo , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , HumanosRESUMO
In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4-7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin's loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Técnicas In Vitro , CoesinasRESUMO
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Trifosfato de Adenosina/química , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Cromossomos/fisiologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Complexos Multiproteicos/fisiologia , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , CoesinasRESUMO
Atomic force microscopy allows visualization of biomolecules with nanometer resolution under physiological conditions. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, meaning that it is now possible to watch single biomolecules in action in real time. Here, we review this development.
Assuntos
Microscopia de Força Atômica/métodos , Nanopartículas/ultraestrutura , Bactérias/ultraestrutura , Células Eucarióticas/ultraestrutura , Nanopartículas/química , Nanotecnologia/métodosRESUMO
Condensin, a key component of the structure maintenance of chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into chromosomes. Here we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these motor proteins, which, individually, extrude DNA in one direction only are able to dynamically change each other's DNA loop sizes, even when far apart. When they are in close proximity, condensin complexes are able to traverse each other and form a loop structure, which we term a Z-loop-three double-stranded DNA helices aligned in parallel with one condensin at each edge. Z-loops can fill gaps left by single loops and can form symmetric dimer motors that pull in DNA from both sides. These findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae , Adenosina Trifosfatases/química , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/química , Cromossomos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Motores Moleculares/química , Complexos Multiproteicos/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Imagem com Lapso de TempoRESUMO
DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.
Assuntos
DNA Super-Helicoidal , DNA , Fluorescência , Substâncias Intercalantes/química , Plasmídeos/genéticaRESUMO
Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.
Assuntos
DNA Bacteriano , DNA Super-Helicoidal , Transcrição Gênica , DNA/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNARESUMO
In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.
Assuntos
Cromossomos Bacterianos , Bactérias/genética , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismoRESUMO
Self-organized pattern formation is vital for many biological processes. Reaction-diffusion models have advanced our understanding of how biological systems develop spatial structures, starting from homogeneity. However, biological processes inherently involve multiple spatial and temporal scales and transition from one pattern to another over time, rather than progressing from homogeneity to a pattern. To deal with such multiscale systems, coarse-graining methods are needed that allow the dynamics to be reduced to the relevant degrees of freedom at large scales, but without losing information about the patterns at small scales. Here, we present a semiphenomenological approach which exploits mass conservation in pattern formation, and enables reconstruction of information about patterns from the large-scale dynamics. The basic idea is to partition the domain into distinct regions (coarse grain) and determine instantaneous dispersion relations in each region, which ultimately inform about local pattern-forming instabilities. We illustrate our approach by studying the Min system, a paradigmatic model for protein pattern formation. By performing simulations, we first show that the Min system produces multiscale patterns in a spatially heterogeneous geometry. This prediction is confirmed experimentally by in vitro reconstitution of the Min system. Using a recently developed theoretical framework for mass-conserving reaction-diffusion systems, we show that the spatiotemporal evolution of the total protein densities on large scales reliably predicts the pattern-forming dynamics. Our approach provides an alternative and versatile theoretical framework for complex systems where analytical coarse-graining methods are not applicable, and can, in principle, be applied to a wide range of systems with an underlying conservation law.
Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas de Escherichia coli , Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Difusão , Proteínas de Escherichia coli/química , Modelos TeóricosRESUMO
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
Assuntos
Análise de Sequência de Proteína/métodos , Imagem Individual de Molécula/métodos , Espectrometria de Massas/métodos , Nanotecnologia , Proteínas/química , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the â¼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with â¼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
Nanopores are versatile single-molecule sensors offering a simple label-free readout with great sensitivity. We recently introduced the nanopore electro-osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.
Assuntos
Nanoporos , Proteínas , DNARESUMO
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Viral/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/metabolismo , Colífagos/química , Colífagos/genética , DNA Viral/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação ProteicaRESUMO
Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/ultraestrutura , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Humanos , Microscopia de Fluorescência , Peptidoglicano/biossíntese , Peptidoglicano/genética , Infecções Pneumocócicas/genética , Streptococcus pneumoniae/patogenicidade , Streptococcus pneumoniae/ultraestruturaRESUMO
Condensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction can proceed in large steps, driving DNA molecules into a fully condensed state against forces of up to 2 pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt insensitive and for the subsequent compaction process. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , DNA Fúngico/química , Proteínas de Ligação a DNA/genética , Hidrólise , Magnetismo , Modelos Moleculares , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Pinças Ópticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Eletricidade EstáticaRESUMO
Holes in metal films do not allow the propagation of light if the wavelength is much larger than the hole diameter, establishing such nanopores as so-called zero-mode waveguides (ZMWs). Molecules, on the other hand, can still pass through these holes. We use this to detect individual fluorophore-labelled molecules as they travel through a ZMW and thereby traverse from the dark region to the illuminated side, upon which they emit fluorescent light. This is beneficial both for background suppression and to prevent premature bleaching. We use palladium as a novel metal-film material for ZMWs, which is advantageous compared to conventionally used metals. We demonstrate that it is possible to simultaneously detect translocations of individual free fluorophores of different colours. Labelled DNA and protein biomolecules can also be detected at the single-molecule level with a high signal-to-noise ratio and at high bandwidth, which opens the door to a variety of single-molecule biophysics studies.
RESUMO
Graphene quantum dots (QDs) are intensively studied as platforms for the next generation of quantum electronic devices. Fine tuning of the transport properties in monolayer graphene QDs, in particular with respect to the independent modulation of the tunnel barrier transparencies, remains challenging and is typically addressed using electrostatic gating. We investigate charge transport in back-gated graphene mechanical break junctions and reveal Coulomb blockade physics characteristic of a single, high-quality QD when a nanogap is opened in a graphene constriction. By mechanically controlling the distance across the newly formed graphene nanogap, we achieve reversible tunability of the tunnel coupling to the drain electrode by 5 orders of magnitude, while keeping the source-QD tunnel coupling constant. The break junction device can therefore become a powerful platform to study the physical parameters that are crucial to the development of future graphene-based devices, including energy converters and quantum calorimeters.
RESUMO
The fine interplay between the simultaneous stretching and confinement of amyloid fibrils is probed by combining a microcapillary setup with atomic force microscopy. Single-molecule statistics reveal how the stretching of fibrils changed from force to confinement dominated at different length scales. System order, however, is solely ruled by confinement. Coarse-grained simulations support the results and display the potential to tailor system properties by tuning the two effects. These findings may further help shed light on in vivo amyloid fibril growth and transport in highly confined environments such as blood vessels.