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Integrating Single-Molecule Visualization and DNA VU Research Portal The DNA double helix challenged by force Gross, P. 2011 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Gross, P. (2011). The DNA double helix challenged by force. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 24. Sep. 2021 The DNA double helix challenged by force This thesis was reviewed by: prof.dr. Ulrich Bockelmann ESPCI CNRS UMR Gulliver, Paris prof.dr. Fred C. MacKintosh VU University, Amsterdam prof.dr. Antoine M. van Oijen University of Groningen prof.dr. Helmut Schiessel Leiden University prof.dr. Mark C. Williams Northeastern University, Boston prof.dr. Gijs J. L. Wuite VU University, Amsterdam This work is part of the research programme ATLAS, a European Commission-funded Marie Curie early stage training network. The research was performed in the section ‘Physics of Complex Systems’ at the VU University, Amsterdam. promotor: prof.dr. G.J.L. Wuite copromotor: dr.ir. E.J.G. Peterman Contents 1 Introduction1 1.1 What is (so excting about) life? ........................ 2 1.2 Cells......................................... 7 1.2.1 What makes cells alive?......................... 8 1.3 DNA, the molecular carrier of life....................... 13 1.3.1 From DNA to proteins (one-way road)................ 17 1.3.2 DNA replication ............................. 23 1.4 Cells tightly control their protein levels.................... 25 1.4.1 Epigenetics ................................ 27 1.4.2 Chromatin................................. 28 1.5 DNA as a physical object............................. 30 1.5.1 DNA as a garden hose.......................... 30 1.6 Outline of this thesis............................... 34 2 Combining optical tweezers and fluorescence microscopy 37 2.1 Introduction .................................... 38 2.2 Instrumentation.................................. 40 2.2.1 Optical Tweezers............................. 41 2.2.2 Fluorescence microscopy........................ 47 2.2.3 Microfluidics ............................... 50 2.3 Preparation of reagents ............................. 53 2.3.1 Terminally-labeled DNA molecules.................. 53 2.3.2 Fluorescent labeling of DNA...................... 54 2.3.3 Fluorescent labeling of proteins.................... 55 2.4 Combining optical trapping, fluorescence microscopy and microfluidics 56 2.4.1 Example protocol II: Sequential isolation and visualization of a single DNA molecule using YOYO-1................. 56 2.4.2 Example protocol III: binding of RPA to ssDNA .......... 58 2.4.3 Example protocol IV: binding or RAD51 to dsDNA . 60 2.5 Conclusions..................................... 61 3 Visualizing the structure of overstretched DNA 63 3.1 Introduction .................................... 64 3.2 Results........................................ 64 3.2.1 DNA overstretching with unconstrained extremities . 66 v CONTENTS 3.2.2 Overstretching torsionally constrained DNA............ 71 3.3 Discussion ..................................... 73 3.4 Methods....................................... 75 4 Twist, stretch and melt: quantifying how DNA complies to tension 79 4.1 Introduction .................................... 80 4.2 Results........................................ 80 4.2.1 Unwinding and overwinding of DNA under tension . 82 4.2.2 Force-induced DNA-strand unpeeling ................ 85 4.2.3 Hysteresis during DNA strand reannealing............. 90 4.3 Discussion ..................................... 92 4.4 Methods....................................... 93 5 Balance between two overstretching modes: melting in the interior vs. from the extremities 95 5.1 Introduction .................................... 96 5.2 Results........................................ 97 5.2.1 An alternative overstretching mechanism in the absence of un- peeling................................... 97 5.2.2 Melting bubbles appear upon unpeeling arrest........... 99 5.2.3 Electrostatic shielding assists melting-bubble formation . 100 5.2.4 Unpeeling stalls in GC-rich regions.................. 102 5.2.5 Melting bubble formation initiates at AT-rich regions . 103 5.2.6 Both melting modes differ significantly when approaching final strand separation............................. 105 5.3 Discussion ..................................... 106 5.4 Methods....................................... 108 6 Melting bubble statistics and dynamics of overstretched DNA 109 6.1 Introduction .................................... 110 6.2 Results........................................ 111 6.2.1 Monitoring the magnitude and the dynamics of fluctuations dur- ing force-induced bubble melting................... 111 6.2.2 Visualizing the number of bubbles.................. 113 6.2.3 Dynamics of melting fluctuations at different stages of the over- stretching transition........................... 115 6.2.4 Modeling DNA fluctuations during DNA overstretching . 115 6.3 Discussion ..................................... 122 vi CONTENTS 6.4 Methods....................................... 124 7 Single-stranded DNA templates for optical tweezers-experiments 127 7.1 Introduction .................................... 128 7.2 Materials and Methods.............................. 129 7.2.1 Biotinylation of two plasmid DNAs (pTR19-ASDS and pKYB1) on both ends of one strand......................... 129 7.2.2 Optical Tweezers Setup......................... 131 7.3 Results........................................ 132 7.3.1 dsDNA denatures at tensions exceeding the overstretching force 132 7.3.2 Dynamics of force-induced DNA denaturation . 134 7.4 Discussion ..................................... 137 List of publications 139 References 141 vii 1 Introduction Making the simple complicated is commonplace; making the complicated simple, awesomely simple, that’s creativity. Charles Mingus Charles Mingus, the famous jazz composer and bass player (1992-1979), is talking about music here, but actually the quote is far more general. Jazz musicians and scientists face some similar challenges. Both the scientist in his laboratory, the jazz musician during a gig, are exposed to significant complexity; nature itself for the sci- entist, or the seemingly effortless interplay of several skilled musicians during a jazz piece. Only the talent to break down all the input into small pieces, seeing structure and patterns in what happens around them enables both, the scientist and the jazz musician to function; channeling the surplus of input into a creative output. Life and thus Biology is, by all means, intrinsically tied to complexity. Scientists study- ing life, mostly unaware of Charles Mingus’ thoughts, still follow his brain wave, re- ducing complexity, studying isolated systems, searching for structure, repetition and general patterns. Even though life comes in a multitude of different appearances, we can find a surprising amount of similarities when we zoom in, and look at how life is organized on the small scale. On the molecular level, organisms that look completely different on the large scale reveal very similar features. One of the most fundamental observations on the molecular level of life is the ubiquitous presence of DNA. Life and DNA are not separable, DNA is essential for life. DNA will be in the focus in this thesis. Since DNA is a vital ingredient of life, I will first try to shed some light on its place in life in this introduction. 1 CHAPTER 1. INTRODUCTION The first question that I want to address in my thesis is maybe the most funda- mental one: Why is it so exciting to study life? Naturally, and in contrast to the rest of this thesis, I will not even attempt to provide an objective view on the first question in this introductory part, since I believe these questions can and should be only addressed in a personal way. 1.1 What is (so excting about) life? Life surrounds us every day. One of the first observations we make really early in our life is that life, and nature, is immensely diverse. An example of this is illustrated in figure 1.1. This diversity of life is one of the most stunning and fascinating facts that makes people go to the zoo, watch wildlife documentaries, and makes little children follow each beetle or butterfly they see. In other words, it naturally brings out the scientist, or the discoverer in us. Life comes in a vast amount of different forms, and continues to surprise us in its creative ability to adapt to such an enormous variety of different environments. This talent to blend into almost every niche on this planet inspired Charles Darwin (1809 - 1882) to formulate his theory of ’the origin of species’ [1]. Darwin was a keen observer and careful analyst. His early interest in science, greatly stimulated by the naturalist
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