DOCSLIB.ORG

A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins molecules Article A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins Roberto Fabian, Jr. 1,*, Santosh Gaire 1, Christopher Tyson 2, Raghabendra Adhikari 3, Ian Pegg 1 and Abhijit Sarkar 1 1 Department of Physics and Vitreous State Laboratory, The Catholic University of America, Washington, DC 20064, USA; [email protected] (S.G.); [email protected] (I.P.); [email protected] (A.S.) 2 Biomedical Engineering Department and Vitreous State Laboratory, The Catholic University of America, Washington, DC 20064, USA; [email protected] 3 Department of Biology, The Catholic University of America, Washington, DC 20064, USA; [email protected] * Correspondence: [email protected] Abstract: We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on Citation: Fabian, R., Jr.; Gaire, S.; force-mediated DNA-core histone assembly and disassembly processes. Tyson, C.; Adhikari, R.; Pegg, I.; Sarkar, A. A Horizontal Magnetic Tweezers for Studying Single DNA Keywords: single molecule micromanipulation techniques; horizontal magnetic tweezers; DNA- Molecules and DNA-Binding histone interactions; nucleosomes Proteins. Molecules 2021, 26, 4781. https://doi.org/10.3390/ molecules26164781 1. Introduction Academic Editor: Francesco Histones are the basic protein unit of the nucleosome core particle. The core particle Simone Ruggeri allows for the first stage of DNA compaction in eukaryotic cells, which involves the formation of a linear array of nucleosomes along the DNA [1]. The four types of histones, Received: 30 June 2021 H2A, H2B, H3, and H4, combine to form an octameric complex comprising two copies of Accepted: 3 August 2021 each type of histone. The interaction between the DNA and the histones are non-specific, Published: 7 August 2021 with the DNA wrapped approximately 1.75 times [2] around the octameric complex. The fifth type of histone, H1 or H5, serves as a link between the adjacent nucleosomes. Publisher’s Note: MDPI stays neutral Electrostatic attraction is responsible for the binding of histones to the DNA. DNA has with regard to jurisdictional claims in a net negative charge on its phosphate backbone, while histones have a net positive charge published maps and institutional affil- in their amino terminal tails, which results in a strong electrostatic attraction. Additionally, iations. the helix dipoles in H2B, H3, and H4 contribute to the interaction with DNA [3]. As a result, the formation of a single nucleosome results in a relatively large decrease in free energy of ~20 kBT[4]. The strong electrostatic interactions between DNA and histones present a great chal- Copyright: © 2021 by the authors. lenge to the cell’s genetic machinery. Genetic processes like DNA transcription, DNA Licensee MDPI, Basel, Switzerland. repair, and DNA replication, which involve specialized protein machinery, require access to This article is an open access article the sections of the DNA stored in the nucleosomes but that is rendered inaccessible by the distributed under the terms and strong binding between the DNA and the histone core particles. The application of force conditions of the Creative Commons and torque by DNA-bound protein complexes may alter the stability of the nucleosome Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and may play a key role in providing access to the DNA. Interestingly, several molecular 4.0/). machines such as RNA polymerase and DNA polymerase have been observed to apply [5] Molecules 2021, 26, 4781. https://doi.org/10.3390/molecules26164781 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4781 2 of 12 large amounts of force in the range of ~10 pN to single DNA templates. The theoretical estimate of the minimum force, f*, needed to mechanically destabilize a nucleosome is ~2 pN [4]. This suggests that in vivo protein machines may be able to apply forces large enough to locally affect the stability of the nucleosomes. Optical traps and magnetic tweezers have been used to investigate force-mediated nucleosome dynamics. Several studies have been performed using different procedures for histone purification and nucleosome reconstitution [6–9]. The results depend on (a) the experimental technique used, i.e., optical or magnetic tweezers, (b) the protocols used for nucleosome reconstitution, (c) the degree of control of the posttranslational modifications, (d) the absence or presence of linker histones, (e) the ionic conditions, (f) the buffer com- positions, and (g) other experimental factors [6]. In Sections 1.1 and 1.2, we summarize results from selected studies on nucleosomes using optical trapping and magnetic tweez- ers and briefly discuss how they have contributed to our understanding of the structure and dynamics of nucleosomes and chromatin. In Section 1.3, we introduce and motivate our study. 1.1. Single Molecule Studies on Nucleosomes Using Optical Traps Optical traps and magnetic tweezers have been used to probe the structure and dynamics of chromatin. In this section, we summarize selected references on the study of single molecule experiments on nucleosomes using optical tweezers. Cui and Bustamante [10] pioneered single molecule micromanipulation studies on nucleosomes using optical tweezers. In their experiments, they stretched and relaxed single chromatin fibers extracted from chicken erythrocytes at three different NaCl concentrations: 5 mM, 40 mM, and 150 mM. Their results established that the force versus extension curves of single chromatin fibers are reversible at forces less than 6 pN and that are irreversible at forces greater than 20 pN in all three NaCl concentrations. Nucleosome disassembly events were not reported in their experiments. Following this study, Bennink et al. [11] performed optical trapping experiments on single chromatin fibers reconstituted from λ-DNA and Xenopus laevis egg extract. They were able to detect nucleosome disruption events starting at 20 pN and up to 40 pN. These were associated with three disruption lengths 65 nm, 130 nm, and 195 nm, which were interpreted as the signatures of one, two, and three nucleosome disassembly events, respectively. Brower-Toland et al. [12] investigated single nucleosomes using optical tweezers with nucleosomes reconstituted from single DNA molecules and core histones (without linker histones) purified using the salt dialysis method. The DNA tethers included 17 tandem repeats of 5S positioning sequences. They found a ~25 nm disruption length of DNA per nucleosome at forces less than 15 pN and ~27 nm disruption lengths for higher forces. Using optical tweezers, Gemmen et al. [13] investigated nucleosome arrays formed from DNA tethers with random sequences, core histones (without linker histones), the histones chaperone NAP1, and the remodeling factor ACF. They reported that the length released per nucleosome unbinding event changed from ~18 nm to ~31 nm from 5 pN to 65 pN. They also found the average force needed to unravel a single nucleosome was dependent on the NaCl concentration of the buffer, which they suggested reflected variations in the binding affinity of the histone core particle to different sequences of the DNA. Claudet et al. [14] used optical tweezers to study nucleosome arrays reconstituted us- ing salt dialysis. A DNA template with 12 tandem repeats of the 5S nucleosome positioning sequence and core histones (without linker histones) was used. They also conducted com- plementary studies using nucleosome arrays extracted from chicken erythrocytes. A total of two DNA disruption lengths of 25 nm and 50 nm were reported in their studies. Disruption events were observed to occur at forces ≥ 20 pN in both types of nucleosome arrays. Pope et al. [15] used an optical tweezers technique to stretch single chromatin fibers assembled from λ-DNA and Xenopus laevis egg extract at different loading rates. They Molecules 2021, 26, 4781 3 of 12 reported three disruption lengths: 30 nm, 59 nm, and 117 nm. All three disruption lengths occurred at forces ≥ 20 pN. Mihardja et al. [16] investigated single mononucleosomal fibers using optical tweez- ers. The mononucleosomal DNA was assembled from 2582 bp DNA containing a single 601 positioning sequences and core histones extracted from chicken erythrocytes. In their experiments, they observed two disruption lengths: 21 nm at forces starting at 3 pN and 22 nm at forces between 8 and 9 pN. They interpreted these as two stages of nucleosome disassembly. The first stage represented the release of the outer wrap of the nucleosomal DNA while the second stage represented the unwrapping of the remaining inner wrap of the nucleosomal DNA. Bussiek et al. [17] studied single chromatin fibers reconstituted from two types of DNA templates, core histones (no linker histones), and the NAP1/ACF system. The two DNA templates were heterogenous DNA, i.e., DNA with a random sequence, and tandem repeat alpha satellite DNA. They extended the individual fibers using optical tweezers. They found that the disruption length in both DNA templates were almost same, ~23 nm for heterogenous DNA and ~24 nm for alpha satellite DNA. However, the disruption force in the alpha satellite DNA was 26 pN, higher than the heterogenous DNA, which showed a disruption force of 22 pN. 1.2. Single Molecule Studies on Nucleosomes Using Magnetic Tweezers Leuba et al. [18] were the first to study nucleosome arrays using vertical magnetic tweezers.
Load more

Recommended publications
  • Datasheet for Histone H2B Human, Recombinant (M2505; Lot 0031404)
    Supplied in: 20 mM Sodium Phosphate (pH 7.0), Quality Control Assays: Ionization-Time of Flight Mass Spectrometry). The Histone H2B 300 mM NaCl and 1 mM EDTA. SDS-PAGE: 0.5 µg, 1.0 µg, 2.0 µg, 5.0 µg, average mass calculated from primary sequence Human, Recombinant 10.0 µg Histone H2B Human, Recombinant were is 13788.97 Da. This confirms the protein identity Note: The protein concentration (1 mg/ml, 73 µM) loaded on a 10–20% Tris-Glycine SDS-PAGE gel as well as the absence of any modifications of the is calculated using the molar extinction coefficient and stained with Coomassie Blue. The calculated histone. 1-800-632-7799 for Histone H2B (6400) and its absorbance at molecular weight is 13788.97 Da. Its apparent [email protected] 280 nm (3,4). 1.0 A units = 2.2 mg/ml N-terminal Protein Sequencing: Protein identity 280 molecular weight on 10–20% Tris-Glycine SDS- www.neb.com was confirmed using Edman Degradation to PAGE gel is ~17 kDa. M2505S 003140416041 Synonyms: Histone H2B/q, Histone H2B.1, sequence the intact protein. Histone H2B-GL105 Mass Spectrometry: The mass of purified Histone H2B Human, Recombinant is 13788.5 Da Protease Assay: After incubation of 10 µg of M2505S B r kDa 1 2 3 4 5 6 7 as determined by ESI-TOF MS (Electrospray Histone H2B Human, Recombinant with a standard 250 4.0 mixture of proteins for 2 hours at 37°C, no 100 µg 1.0 mg/ml Lot: 0031404 150 13788.5 100 proteolytic activity could be detected by SDS- RECOMBINANT Store at –20°C Exp: 4/16 80 PAGE.
    [Show full text]
  • A-10 Optical and Magnetic Tweezers
    A-10 Optical and Magnetic Tweezers From Physics 191r Optical Tweezer Word version File:A10tweezer 10.doc Optical Tweezer PDF version File:A10tweezer 10.pdf ELECTROMAGNETIC TRAPPING: Optical and Magnetic Tweezers author: Mara Prentiss, 2004 first experiment: yes Optical tweezer video (http://www.fas.harvard.edu/~phys191r/Bench_Notes/op_tweez.mov) Magnetic tweezer video (http://www.fas.harvard.edu/~phys191r/Bench_Notes/mag_tweez.mov) Contents 1 LEARNING GOALS 2 INTRODUCTION 3 APPARATUS 3.1 Laser 3.2 Laser controller 3.3 Laser power monitor 3.4 Laser beam profiler 3.5 Sample Preparation 3.5.1 Optical Tweezer 3.5.2 Magnetic Tweezer 3.6 Copper sample stage with temperature control 3.7 Translation stage and controller 3.8 Function generator/circle box/oscilloscope 3.9 White light source 3.10 Camera 3.11 Microspheres 3.12 Quadrant photodiode with I/V converter 3.13 Magnetic Tweezer 4 EXPERIMENTAL PROCEDURE 4.1 Investigations of the Optical Trap 4.2 Investigations of Thermal Motion 4.3 Optional Optical Tweezer Experiments 4.4 Interparticle Interaction 4.5 Magnetic Tweezer 5 NOTES 6 REFERENCES 6.1 Optical Traps 6.2 Brownian Motion 6.3 Magnetic Field Gradient Traps 6.4 2D Polymer Sphere Crystals 6.5 2D Melting 6.6 Ordered Trapped Particles 6.7 Electromagnetic Traps 7 BENCH NOTES 8 APPENDIX A: Optical Tweezer Alignment Procedure 8.1 Safety 8.2 Beam alignment 8.3 Camera alignment 8.4 Trapping a particle 9 APPENDIX B: Particle Tracking using IDL 10 APPENDIX C: Particle Tracking using MATLAB 10.1 Purpose 10.2 Outline 10.3 Details 10.3.1 1) Make videos 10.3.2 2) Import video and convert to grayscale 10.3.3 3) Analyze the videos 10.3.4 4) Analyze the data files LEARNING GOALS Trap uncharged dielectric microbeads in aqueous solution with a focused laser beam.
    [Show full text]
  • Topography of the Histone Octamer Surface: Repeating Structural Motifs Utilized in the Docking of Nucleosomal
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10489-10493, November 1993 Biochemistry Topography of the histone octamer surface: Repeating structural motifs utilized in the docking of nucleosomal DNA (histone fold/helix-strand-helix motif/parallel fi bridge/binary DNA binding sites/nucleosome) GINA ARENTS* AND EVANGELOS N. MOUDRIANAKIS*t *Department of Biology, The Johns Hopkins University, Baltimore, MD 21218; and tDepartment of Biology, University of Athens, Athens, Greece Communicated by Christian B. Anfinsen, August 5, 1993 ABSTRACT The histone octamer core of the nucleosome is interactions. The model offers strong predictive criteria for a protein superhelix offour spirally arrayed histone dimers. The structural and genetic biology. cylindrical face of this superhelix is marked by intradimer and interdimer pseudodyad axes, which derive from the nature ofthe METHODS histone fold. The histone fold appears as the result of a tandem, parallel duplication of the "helix-strand-helix" motif. This The determination of the structure of the histone octamer at motif, by its occurrence in the four dimers, gives rise torepetitive 3.1 A has been described (3). The overall shape and volume structural elements-i.e., the "parallel 13 bridges" and the of this tripartite structure is in agreement with the results of "paired ends of helix I" motifs. A preponderance of positive three independent studies based on differing methodolo- charges on the surface of the octamer appears as a left-handed gies-i.e., x-ray diffraction, neutron diffraction, and electron spiral situated at the expected path of the DNA. We have microscopic image reconstruction (4-6). Furthermore, the matched a subset of DNA pseudodyads with the octamer identification of the histone fold, a tertiary structure motif of pseudodyads and thus have built a model of the nucleosome.
    [Show full text]
  • Invincible DNA Tethers: Covalent DNA Anchoring for Enhanced Temporal and Force Stability in Magnetic Tweezers Experiments Richard Janissen, Bojk A
    Nucleic Acids Research Advance Access published August 19, 2014 Nucleic Acids Research, 2014 1 doi: 10.1093/nar/gku677 Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments Richard Janissen, Bojk A. Berghuis, David Dulin, Max Wink, Theo van Laar and Nynke H. Dekker* Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands Received May 30, 2014; Revised July 11, 2014; Accepted July 14, 2014 ABSTRACT mental role in a variety of biological processes ranging from cellular motility to the replication, repair and segregation of Downloaded from Magnetic tweezers are a powerful single-molecule DNA (1,2). Examples of single-molecule methods capable technique that allows real-time quantitative inves- of applying force to biological systems include atomic force tigation of biomolecular processes under applied spectroscopy (AFM), optical tweezers and magnetic tweez- force. High pulling forces exceeding tens of picoNew- ers (2,3). The magnetic and optical tweezers methods have tons may be required, e.g. to probe the force range garnered particular attention as they provide high tempo- http://nar.oxfordjournals.org/ of proteins that actively transcribe or package the ral, spatial and force sensitivity in a range from hundreds of genome. Frequently, however, the application of such femtoNewtons to tens of picoNewtons (pN), and, in con- forces decreases the sample lifetime, hindering data trast to AFM, they are also capable of measuring and ap- acquisition. To provide experimentally viable sam- plying torque (4–7). More recent developments in magnetic ple lifetimes in the face of high pulling forces, we tweezers also include the possibility of measuring multiple samples simultaneously (multiplexing) (8–10), allowing for have designed a novel anchoring strategy for DNA a broad, statistically sound characterization of biomolecu- in magnetic tweezers.
    [Show full text]
  • Building Bridges Within the Bacterial Chromosome
    Review Building bridges within the bacterial chromosome 1,2 2 Dan Song and Joseph J. Loparo 1 Harvard Biophysics Program, Harvard Medical School, Boston, MA, USA 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA All organisms must dramatically compact their genomes called macrodomains (Box 1) with a size of approximately to accommodate DNA within the cell. Bacteria use a set 1 Mb [2,3]. of DNA-binding proteins with low sequence specificity For the past two decades, studies on bacterial chromo- called nucleoid-associated proteins (NAPs) to assist in some condensation and organization have focused on NAPs chromosome condensation and organization. By bend- that can be classified as DNA benders or DNA bridgers ing or bridging DNA, NAPs also facilitate chromosome (Figure 1). Examples of well-studied DNA benders include segregation and regulate gene expression. Over the past HU (a histone-like protein from Escherichia coli strain U93) decade, emerging single-molecule and chromosome and integration host factor (IHF). These proteins can non- conformation capture techniques have investigated specifically bind to DNA by inserting two arms into the minor the molecular mechanisms by which NAPs remodel groove, inducing curvature of more than 908 [4]. DNA and organize the bacterial chromosome. In this review bridgers form dynamic connections between DNA duplexes we describe how such approaches reveal the biochemi- resulting in large DNA loops that can span several kilobases. cal mechanisms of three NAPs that are believed to Besides condensing chromosomes, DNA bridgers also func- facilitate DNA bridging: histone-like nucleoid structuring tion in chromosome segregation and transcription silencing.
    [Show full text]
  • Tissue Manipulation Using Nano-Particles Ferrofluids for Minimal Access Surgical Applications
    Tissue Manipulation Using Nano-Particles Ferrofluids for Minimal Access Surgical Applications YU-SHENG LIN Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Mechanical Engineering September, 2014 The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. - ii - Acknowledgements I would like to express my deepest gratitude to my Ph.D. supervisor, Professor Anne Neville for her constant support and guidance through my Ph.D. study. Her enthusiasm, the effort she put into the research and her patient all together make this thesis possible. I am grateful to thank two second supervisors, Dr Rupesh Roshan and Dr Tomasz Liskiewicz, who had supported me in daily research and discussions. I also would like to thank all who had helped this work develop well: the great clinical support from Professor David Jayne, Adrian Hood and Jenifer Barrie on the in-vivo study; the great technical support from Dr Peter Culmer and Dr Ali Alazmani in designing the ex-vivo experimental setup using LabVIEW; the technical support from Dr Nagitha Wijayathunga in characterising the particle migration using micro- CT scanner; the great support from Dr Yi-Fan Chou in processing images using Matlab. In addition, I would like to thank my two examiners, Professor Peter Brett and Dr Mark Wilson for the advices to make this thesis become better.
    [Show full text]
  • A Mutation in Histone H2B Represents a New Class of Oncogenic Driver
    Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/2159-8290.CD-19-0393 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver Richard L. Bennett1, Aditya Bele1, Eliza C. Small2, Christine M. Will2, Behnam Nabet3, Jon A. Oyer2, Xiaoxiao Huang1,9, Rajarshi P. Ghosh4, Adrian T. Grzybowski5, Tao Yu6, Qiao Zhang7, Alberto Riva8, Tanmay P. Lele7, George C. Schatz9, Neil L. Kelleher9 Alexander J. Ruthenburg5, Jan Liphardt4 and Jonathan D. Licht1 * 1 Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, FL 2 Division of Hematology/Oncology, Northwestern University 3 Department of Cancer Biology, Dana Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School 4 Department of Bioengineering, Stanford University 5 Department of Molecular Genetics and Cell Biology, The University of Chicago 6 Department of Chemistry, Tennessee Technological University 7 Department of Chemical Engineering, University of Florida 8 Bioinformatics Core, Interdisciplinary Center for Biotechnology Research, University of Florida 9 Department of Chemistry, Northwestern University, Evanston IL 60208 Running title: Histone mutations in cancer *Corresponding Author: Jonathan D. Licht, MD The University of Florida Health Cancer Center Cancer and Genetics Research Complex, Suite 145 2033 Mowry Road Gainesville, FL 32610 352-273-8143 [email protected] Disclosures: The authors have no conflicts of interest to declare Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/2159-8290.CD-19-0393 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
    [Show full text]
  • Tuning Sizes, Morphologies, and Magnetic Properties of Monocore
    Tuning Sizes, Morphologies, and Magnetic Properties of Monocore Versus Multicore Iron Oxide Nanoparticles through the Controlled Addition of Water in the Polyol Synthesis Gauvin Hémery, Anthony C. Keyes Jr, Eneko Garaio, Irati Rodrigo, Jose Angel Garcia, Fernando Plazaola, Elisabeth Garanger, Olivier Sandre To cite this version: Gauvin Hémery, Anthony C. Keyes Jr, Eneko Garaio, Irati Rodrigo, Jose Angel Garcia, et al.. Tuning Sizes, Morphologies, and Magnetic Properties of Monocore Versus Multicore Iron Oxide Nanoparticles through the Controlled Addition of Water in the Polyol Synthesis. Inorganic Chemistry, American Chemical Society, 2017, 56 (14), pp.8232-8243. 10.1021/acs.inorgchem.7b00956. hal-01567664v3 HAL Id: hal-01567664 https://hal.archives-ouvertes.fr/hal-01567664v3 Submitted on 17 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives| 4.0 International License This document is the author manuscript version of published paper Inorganic Chemistry, 2017, 56(14), 8232−8243, after peer review and technical editing by the publisher. Final version visible on DOI:10.1021/acs.inorgchem.7b00956 Tuning Sizes, Morphologies, and Magnetic Properties of Monocore Versus Multicore Iron Oxide Nanoparticles through the Controlled Addition of Water in the Polyol Synthesis Gauvin Hemerya, Anthony C.
    [Show full text]
  • Chromatin Structure
    Chromatin Structure Dr. Carol S. Newlon [email protected] ICPH E250P DNA Packaging Is a Formidable Challenge • Single DNA molecule in human chromosome ca. 5 cm long • Diploid genome contains ca. 2 meters of DNA • Nucleus of human cell ca. 5 µm in diameter • Human metaphase chromosome ca. 2.5 µm in length • 10,000 to 20,000 packaging ratio required Overview of DNA Packaging Packaging in Interphase Nucleus Chromatin Composition • Complex of DNA and histones in 1:1 mass ratio • Histones are small basic proteins – highly conserved during evolution – abundance of positively charged aa’s (lysine and arginine) bind negatively charged DNA • Four core histones: H2A, H2B, H3, H4 in 1:1:1:1 ratio • Linker histone: H1 in variable ratio Chromatin Fibers 11-nm fiber 30-nm fiber • beads = nucleosomes • physiological ionic • compaction = 2.5X strength (0.15 M KCl) • low ionic strength buffer • compaction = 42X • H1 not required • H1 required Micrococcal Nuclease Digestion of Chromatin Stochiometry of Histones and DNA • 146 bp DNA ca. 100 kDa • 8 histones ca 108 kDa • mass ratio of DNA:protein 1:1 Structure of Core Nucleosome 1.65 left handed turns of DNA around histone octamer Histone Structure Assembly of a Histone Octamer Nucleosomes Are Dynamic Chromatin Remodeling Large complexes of ≥ 10 proteins Use energy of ATP hydrolysis to partially disrupt histone-DNA contacts Catalyze nucleosome sliding or nucleosome removal 30-nm Chromatin Fiber Structure ?? Models for H1 and Core Histone Tails in Formation of 30-nm Fiber Histone Tails Covalent Modifications
    [Show full text]
  • The Catholic University of America
    THE CATHOLIC UNIVERSITY OF AMERICA Development and Use of Novel Transverse Magnetic Tweezers for Single-Molecule Studies of DNA-Protein Interactions A DISSERTATION Submitted to the Faculty of the Department of Biomedical Engineering School of Engineering Of The Catholic University of America In Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy By Christopher D. Tyson Washington, D.C. 2016 Development and Use of Novel Transverse Magnetic Tweezers for Single-Molecule Studies of DNA-Protein Interactions Christopher D. Tyson, Ph.D. Director: Abhijit Sarkar, Ph.D. I describe several contributions to single molecule experiments. A transverse magnetic tweezers is presented that enables in-plane micromechanical manipulation of a single DNA molecule. This includes a new method for tethering DNA utilizing two labeled beads and a functionalized glass micro-rod. The attachment chemistry reported here enables rapid capture of multiple DNA tethers in parallel, overcomes the difficulties associated with bead aspiration, and preserves the ability to perform differential extension measurements from the bead centroids. Combined with micro- injection pipettes, a new sample cell design, and a buffer exchange system, the components increase the ease-of-use and experimental throughput of the magnetic tweezers device. On the software side, several unique computational methods for interrogating single molecule data are described. First, a technique that uses the diffraction pattern of beads to perform sub-pixel, ~10 nm-level localization of the bead centroids is explained. Second, a novel method for automatically detecting steps in DNA extension data is presented. This algorithm is well-suited for analyzing experiments involving binding and force-induced unbinding of DNA-protein complexes, which produce flat extension regions – steps – corresponding to the times between individual protein association or dissociation events.
    [Show full text]
  • Advances in Magnetic Tweezers for Single Molecule and Cell Biophysics
    FRONTIER REVIEW Advances in Magnetic Tweezers for Single Molecule and Cell Biophysics Devrim Kilinc a and Gil U. Lee* a Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x 5 Magnetic tweezers (MTW) enable highly accurate forces to be transduced to molecules to study mechanotransduction at the molecular or cellular level. We review recent MTW studies in single molecule and cell biophysics that demonstrate the flexibility of this technique. We also discuss technical advances in the method on several fronts, i.e., from novel approaches for the measurement of torque to multiplexed biophysical assays. Finally, we describe multi-component nanorods with enhanced optical 10 and magnetic properties and discuss their potential as future MTW probes. Mechanical manipulation of biological material, from proteins to tissue, is receiving growing interest in biomedical research since it provides quantitative information on how mechanical stimuli regulate a multitude of biological processes from enzyme activity to organ development 1. Among the techniques developed to apply force to biological entities, magnetic tweezers (MTW) has claimed particular attention as it is capable of applying torque, it can be fully integrated with a range of powerful light microscopy imaging modes, and it 2 15 offers high force, spatial, and temporal sensitivity . In this review, we present a brief summary of recent measurements made with MTW and highlight several technical advances that are taking place in MTW instrumentation. The MTW technique is based on the non-invasive manipulation of magnetic particles, and thereby the biological entity they are attached to, via an externally-imposed magnetic field and/or field gradient.
    [Show full text]
  • Magnetic Tweezers: Micromanipulation and Force Measurement at the Molecular Level
    3314 Biophysical Journal Volume 82 June 2002 3314–3329 Magnetic Tweezers: Micromanipulation and Force Measurement at the Molecular Level Charlie Gosse and Vincent Croquette Laboratoire de Physique Statistique, E´ cole Normale Supe´rieure, Unite´ de Recherche 8550 associe´e au Centre National de la Recherche Scientifique et aux Universite´s Paris VI et VII, 75231 Paris, France ABSTRACT Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness ϳ10Ϫ7 N/m. Thus, they can be manipulated in three dimensions at a speed of ϳ10 ␮m/s and rotated along the optical axis at a frequency of 10 Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50 fN to 20 pN. Similarly, nearly horizontal forces up to 5 pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators. ⌫ LIST OF SYMBOLS x,y (nondimensional) the viscous coefficient of a sphere moving parallel to a sidewall (Defined in Ϫ1 Au (NA ) the factor between the force and the cur- Eq.
    [Show full text]