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1.
Dev Cell ; 53(6): 706-723.e5, 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32504558

RESUMO

The Bloom's helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

2.
J Am Chem Soc ; 142(26): 11451-11463, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32496760

RESUMO

Toehold-mediated strand displacement is the most abundantly used method to achieve dynamic switching in DNA-based nanotechnology. An "invader" strand binds to the "toehold" overhang of a target strand and replaces a target-bound "incumbent" strand. Here, the complementarity of the invader to the single-stranded toehold provides the free energy bias of the reaction. Despite the widespread use of strand displacement reactions for realizing dynamic DNA nanostructures, variants on the basic motif have not been completely characterized. Here we introduce a simple thermodynamic model, which is capable of quantitatively describing the kinetics of strand displacement reactions in the presence of mismatches, using a minimal set of parameters. Furthermore, our model highlights that base pair fraying and internal loop formation are important mechanisms when involving mismatches in the displacement process. Our model should provide a helpful tool for the rational design of strand-displacement reaction networks.

3.
Proc Natl Acad Sci U S A ; 117(16): 8859-8869, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32241893

RESUMO

To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.


Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Hidrolases Anidrido Ácido/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos , Hidrólise , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Células Sf9
4.
PLoS One ; 14(11): e0224936, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31747397

RESUMO

The restricted access of regulatory factors to their binding sites on DNA wrapped around the nucleosomes is generally interpreted in terms of molecular shielding exerted by nucleosomal structure and internucleosomal interactions. Binding of proteins to DNA often includes intercalation of hydrophobic amino acids into the DNA. To assess the role of constrained superhelicity in limiting these interactions, we studied the binding of small molecule intercalators to chromatin in close to native conditions by laser scanning cytometry. We demonstrate that the nucleosome-constrained superhelical configuration of DNA is the main barrier to intercalation. As a result, intercalating compounds are virtually excluded from the nucleosome-occupied regions of the chromatin. Binding of intercalators to extranucleosomal regions is limited to a smaller degree, in line with the existence of net supercoiling in the regions comprising linker and nucleosome free DNA. Its relaxation by inducing as few as a single nick per ~50 kb increases intercalation in the entire chromatin loop, demonstrating the possibility for long-distance effects of regulatory potential.


Assuntos
Cromatina/química , DNA/química , Substâncias Intercalantes/farmacologia , Conformação de Ácido Nucleico , Bibliotecas de Moléculas Pequenas/farmacologia , Membrana Celular/metabolismo , Etídio/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Nucleossomos/química , Transcrição Genética
5.
Cell Rep ; 28(12): 3157-3166.e4, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533038

RESUMO

The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems.

6.
EMBO J ; 38(13): e101516, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268598

RESUMO

DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.


Assuntos
DNA/metabolismo , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
7.
Chemistry ; 25(38): 9012-9016, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31081977

RESUMO

DNA nanostructures provide a powerful platform for the programmable assembly of nanomaterials. Here, this approach is extended to semiconductor nanorods that possess interesting electrical properties and could be utilized for the bottom-up fabrication of nanoelectronic building blocks. The assembly scheme is based on an efficient DNA functionalization of the nanorods. A complete coverage of the rod surface with DNA ensures a high colloidal stability while maintaining the rod size and shape. It furthermore supports the assembly of the nanorods at defined docking positions of a DNA origami platform with binding efficiencies of up to 90 % as well as the formation of nanorod dimers with defined relative orientations. By incorporating orthogonal binding sites for gold nanoparticles, defined metal-semiconductor heterostructures can be fabricated. Subsequent application of a seeded growth procedure onto the gold nanoparticles (AuNPs) allows for to establish a direct metal-semiconductor interface as a crucial basis for the integration of semiconductors in self-assembled nanoelectronic devices.

8.
Biophys J ; 116(8): 1394-1405, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30954211

RESUMO

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.


Assuntos
DNA/química , Etídio/análogos & derivados , Substâncias Intercalantes/química , Sítios de Ligação , Fenômenos Biofísicos , Etídio/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Termodinâmica
9.
Nano Lett ; 19(4): 2707-2714, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30887810

RESUMO

Recently introduced DNA nanomolds allow the shape-controlled growth of metallic nanoparticles. Here we demonstrate that this approach can be used to fabricate longer linear metal nanostructures of controlled lengths and patterns. To this end, we establish a set of different interfaces that enable mold interactions with high affinity and specificity. These interfaces enable and control the modular assembly of mold monomers into larger mold superstructure with programmable dimension in which each mold monomer remains uniquely addressable. Preloading the molds with nanoparticle seeds subsequently allows the growth of linear gold nanostructures whose lengths are controlled by the DNA structure. Exploiting the addressability of individual mold monomers furthermore allows achievement of site-specific metallization, that is, to create defined metal patterns. We think that the introduced approach provides a useful basis to fabricate nanomaterials with complex shapes and material composition in a fully programmable and modular fashion.


Assuntos
DNA/química , Fungos/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Ouro/química
10.
PLoS One ; 13(5): e0197659, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29791485

RESUMO

The cellular prion protein (PrPC) is implicated in neuroprotective signaling and neurotoxic pathways in both prion diseases and Alzheimer's disease (AD). Specifically, the intrinsically disordered N-terminal domain (N-PrP) has been shown to interact with neurotoxic ligands, such as Aß and Scrapie prion protein (PrPSc), and to be crucial for the neuroprotective activity of PrPC. To gain further insight into cellular pathways tied to PrP, we analyzed the brain interactome of N-PrP. As a novel approach employing recombinantly expressed PrP and intein-mediated protein ligation, we used N-PrP covalently coupled to beads as a bait for affinity purification. N-PrP beads were incubated with human AD or control brain lysates. N-PrP binding partners were then identified by electrospray ionization tandem mass spectrometry (nano ESI-MS/MS). In addition to newly identified proteins we found many previously described PrP interactors, indicating a crucial role of the intrinsically disordered part of PrP in mediating protein interactions. Moreover, some interactors were found only in either non-AD or AD brain, suggesting aberrant PrPC interactions in the pathogenesis of AD.


Assuntos
Doença de Alzheimer/metabolismo , Proteínas PrPC/metabolismo , Resinas Acrílicas , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis , Domínios e Motivos de Interação entre Proteínas , Espectrometria de Massas por Ionização por Electrospray
11.
J Biol Chem ; 293(21): 8020-8031, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29636413

RESUMO

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.


Assuntos
Neuroblastoma/prevenção & controle , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Multimerização Proteica , Scrapie/prevenção & controle , Animais , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Transporte Proteico , Scrapie/patologia , Células Tumorais Cultivadas
12.
Nucleic Acids Res ; 46(8): 4087-4098, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29596641

RESUMO

In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade-crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Proteínas Associadas a CRISPR/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , Escherichia coli/metabolismo , Mutação , Conformação Proteica
13.
Nano Lett ; 18(3): 2116-2123, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29482327

RESUMO

We introduce a new concept for the solution-based fabrication of conductive gold nanowires using DNA templates. To this end, we employ DNA nanomolds, inside which electroless gold deposition is initiated by site-specific attached seeds. Using programmable interfaces, individual molds self-assemble into micrometer-long mold superstructures. During subsequent internal gold deposition, the mold walls constrain the metal growth, such that highly homogeneous nanowires with 20-30 nm diameters are obtained. Wire contacting using electron-beam lithography and electrical conductance characterization at temperatures between 4.2 K and room temperature demonstrate that metallic conducting wires were produced, although for part of the wires, the conductance is limited by boundaries between gold grains. Using different mold designs, our synthesis scheme will, in the future, allow the fabrication of complex metal structures with programmable shapes.


Assuntos
DNA/química , Condutividade Elétrica , Ouro/química , Nanotecnologia/métodos , Nanofios/química , Nanofios/ultraestrutura , Temperatura
14.
Nucleic Acids Res ; 45(10): 5968-5979, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28453854

RESUMO

Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5΄-CC/SGG-3΄ (where S = C or G, '/' designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level. We find, using FRET, that BcnI adopts either 'open' or 'closed' conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI-DNA interactions.


Assuntos
DNA Viral/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Bacillus/enzimologia , Bacillus/genética , Bacteriófago T7/genética , Domínio Catalítico , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pinças Ópticas , Ligação Proteica , Conformação Proteica , Pontos Quânticos , Especificidade por Substrato
15.
Elife ; 52016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27612385

RESUMO

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.


Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , RecQ Helicases/metabolismo , Helicase da Síndrome de Werner/metabolismo , Humanos , Multimerização Proteica
16.
Methods ; 108: 4-13, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27402355

RESUMO

Magnetic tweezers provide a versatile toolkit supporting the mechanistic investigation of helicases. In the present article, we show that custom magnetic tweezers setups are straightforward to construct and can easily be extended to provide adaptable platforms, capable of addressing a multitude of enquiries regarding the functions of these fascinating molecular machines. We first address the fundamental components of a basic magnetic tweezers scheme and review some previous results to demonstrate the versatility of this instrument. We then elaborate on several extensions to the basic magnetic tweezers scheme, and demonstrate their applications with data from ongoing research. As our methodological overview illustrates, magnetic tweezers are an extremely useful tool for the characterization of helicases and a custom built instrument can be specifically tailored to suit the experimenter's needs.


Assuntos
DNA Helicases/química , Magnetismo , Nanotecnologia/métodos , DNA Helicases/genética , Pinças Ópticas
17.
PLoS One ; 11(5): e0156098, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27214207

RESUMO

In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure.


Assuntos
Proteínas Arqueais/metabolismo , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Pyrococcus furiosus , Proteínas Arqueais/química , Proteínas Arqueais/genética , Células Cultivadas , Clonagem Molecular , DNA/química , DNA Arqueal/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Conformação de Ácido Nucleico , Ligação Proteica , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo
18.
Nucleic Acids Res ; 44(12): 5837-48, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27016742

RESUMO

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg(2+) concentrations, such that human RPA can melt DNA in absence of force.


Assuntos
Replicação do DNA , DNA de Cadeia Simples/genética , Mecanotransdução Celular , Proteína de Replicação A/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fenômenos Biomecânicos , Clonagem Molecular , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Sequências Repetidas Invertidas , Magnésio/metabolismo , Campos Magnéticos , Desnaturação de Ácido Nucleico , Pinças Ópticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tensão Superficial
19.
Nano Lett ; 16(1): 381-6, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26632021

RESUMO

We present a hybrid single-molecule technique combining magnetic tweezers and Förster resonance energy transfer (FRET) measurements. Through applying external forces to a paramagnetic sphere, we induce conformational changes in DNA nanostructures, which are detected in two output channels simultaneously. First, by tracking a magnetic bead with high spatial and temporal resolution, we observe overall DNA length changes along the force axis. Second, the measured FRET efficiency between two fluorescent probes monitors local conformational changes. The synchronized orthogonal readout in different observation channels will facilitate deciphering the complex mechanisms of biomolecular machines.


Assuntos
DNA/química , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Magnetismo , Pinças Ópticas
20.
Biophys J ; 108(10): 2550-2561, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25992733

RESUMO

Magnetic tweezers are a wide-spread tool used to study the mechanics and the function of a large variety of biomolecules and biomolecular machines. This tool uses a magnetic particle and a strong magnetic field gradient to apply defined forces to the molecule of interest. Forces are typically quantified by analyzing the lateral fluctuations of the biomolecule-tethered particle in the direction perpendicular to the applied force. Since the magnetic field pins the anisotropy axis of the particle, the lateral fluctuations follow the geometry of a pendulum with a short pendulum length along and a long pendulum length perpendicular to the field lines. Typically, the short pendulum geometry is used for force calibration by power-spectral-density (PSD) analysis, because the movement of the bead in this direction can be approximated by a simple translational motion. Here, we provide a detailed analysis of the fluctuations according to the long pendulum geometry and show that for this direction, both the translational and the rotational motions of the particle have to be considered. We provide analytical formulas for the PSD of this coupled system that agree well with PSDs obtained in experiments and simulations and that finally allow a faithful quantification of the magnetic force for the long pendulum geometry. We furthermore demonstrate that this methodology allows the calibration of much larger forces than the short pendulum geometry in a tether-length-dependent manner. In addition, the accuracy of determination of the absolute force is improved. Our force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.


Assuntos
Algoritmos , DNA/química , Magnetismo/normas , Calibragem , Magnetismo/métodos , Microfluídica/métodos , Microfluídica/normas
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