RESUMO
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
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
Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3' telomeric overhangs, indicating that MAD2L2 inhibits 5' end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5' end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis. Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5' end resection downstream of RIF1.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Mad2/metabolismo , Reparo de DNA por Recombinação , Telômero/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA por Junção de Extremidades/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/genética , Proteínas Repressoras , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.
Assuntos
Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , DNA/isolamento & purificação , Nanoporos , Sítios de Ligação , DNA/química , DNA/genética , Humanos , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genéticaRESUMO
While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates.
Assuntos
DNA/química , Grafite/química , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Torção Mecânica , DNA/análise , DNA/ultraestrutura , Grafite/análise , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão/métodos , Microscopia Eletrônica de Transmissão/tendências , Nanocompostos/análise , Nanocompostos/química , Nanocompostos/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Nanotecnologia/tendências , Conformação de Ácido NucleicoRESUMO
DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization of supercoiled DNA molecules, named ISD (Intercalation-induced Supercoiling of DNA). We use an intercalating dye to induce supercoiling of surface-attached DNA molecules as well as to visualize coiled-loop structures (i.e., plectonemes) formed on DNA. The technique is solely based on epifluorescence microscopy and requires no mechanical manipulation of the DNA molecules. This new assay allows to track positions and sizes of individual plectonemes and characterize their position-dependent dynamics such as nucleation, termination, and diffusion. We describe the ISD technique and demonstrate its potential by establishing that plectonemes are pinned to a local 10-nucleotide long mispaired sequence along a double-stranded DNA molecule.
Assuntos
DNA Super-Helicoidal/química , Fluorescência , Difusão , Conformação de Ácido NucleicoRESUMO
Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.
Assuntos
Ciclo Celular , Hematopoese , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Anemia/enzimologia , Animais , Apoptose , Biocatálise , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histona Desacetilase 1/deficiência , Histona Desacetilase 2/deficiência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Trombocitopenia/enzimologia , Trombocitopenia/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA por Junção de Extremidades , Transdução de Sinais , Proteína 28 com Motivo Tripartido , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Ubiquitinação , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Fosforilação , Proteína 28 com Motivo Tripartido/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Células HEK293 , Telômero/metabolismo , Dano ao DNA , Cromatina/metabolismo , AnimaisRESUMO
DNA loop extrusion by structural-maintenance-of-chromosome (SMC) complexes has emerged as a primary organizing principle for chromosomes. The mechanism by which SMC motor proteins extrude DNA loops is still unresolved and much debated. The ring-like structure of SMC complexes prompted multiple models where the extruded DNA is topologically or pseudotopologically entrapped within the ring during loop extrusion. However, recent experiments showed the passage of roadblocks much bigger than the SMC ring size, suggesting a nontopological mechanism. Recently, attempts were made to reconcile the observed passage of large roadblocks with a pseudotopological mechanism. Here we examine the predictions of these pseudotopological models and find that they are not consistent with new experimental data on SMC roadblock encounters. Particularly, these models predict the formation of two loops and that roadblocks will reside near the stem of the loop upon encounter-both in contrast to experimental observations. Overall, the experimental data reinforce the notion of a nontopological mechanism for extrusion of DNA.
Assuntos
Cromossomos , DNA , Cromossomos/metabolismo , DNA/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.
Assuntos
Adenosina Trifosfatases , DNA Super-Helicoidal , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA , Complexos Multiproteicos/químicaRESUMO
Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Amplificação de Ácido Nucleico , DNA , Testes Imediatos , RNA Guia de CinetoplastídeosRESUMO
Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97%-100% of DNA-binding proteins are removed from the sample. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labeled DNA locus. These results indicate the practical feasibility of a "genome-in-a-box" approach to study chromosome organization from the bottom up.
Assuntos
Cromossomos , DNA , DNA/genética , Cromossomos/metabolismo , Genoma , Proteínas de Ligação a DNA/genética , Microscopia de FluorescênciaRESUMO
Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.
Assuntos
Nanopartículas , Nucleossomos , Proteínas de Ciclo Celular , Cromatina , Saccharomyces cerevisiaeRESUMO
The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Ligação ProteicaRESUMO
The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.
Assuntos
Reparo do DNA por Junção de Extremidades , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Repressoras/metabolismo , Telômero/metabolismo , Animais , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Camundongos Knockout , Interferência de RNA , Proteínas Repressoras/genética , Telômero/genéticaRESUMO
The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentally confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.
Assuntos
DNA Bacteriano/química , DNA Super-Helicoidal/química , Plasmídeos/química , Sequência de Bases , Biotina/química , Carbocianinas/química , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Corantes Fluorescentes/química , Microscopia de Fluorescência , Compostos Orgânicos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estreptavidina/químicaRESUMO
The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.
Assuntos
DNA/química , Proteínas Intrinsicamente Desordenadas/química , Poro Nuclear/química , Conformação de Ácido Nucleico , Íons , Simulação de Dinâmica Molecular , Nanoporos , Poro Nuclear/ultraestruturaRESUMO
Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo.