University of Groningen
Assembly dynamics of supramolecular protein-DNA complexes studied by single-molecule
fluorescence microscopy
Stratmann, Sarah
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Publication date: 2017
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Stratmann, S. (2017). Assembly dynamics of supramolecular protein-DNA complexes studied by single-molecule fluorescence microscopy. Rijksuniversiteit Groningen.
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Assembly dynamics of
supramolecular protein-DNA
complexes studied by single-
molecule fluorescence microscopy
The work published in this thesis was carried out in the research group Single Molecule Bio-physics at the Zernike Institute for Advanced Materials (ZIAM) of the University of Gron-ingen, The Netherlands. The research was financially supported by the Netherlands Organ-isation for Scientific Research (NWO), European Research Council (ERC) and the Zernike Institute for Advanced Materials.
Copyright © 2017 Sarah Stratmann
All rights reserved. No part of this publication may be produced, stored in a retrieval system of any nature, or transmitted in any form or by any means, electronic, mechanical, including photocopying and recording, without prior written permission of the author.
Printed by: Ipskamp Printing, Enschede
Cover design and layout: Sarah Stratmann & Bastian Niebel ISBN: 978-90-367-9496-1 (printed version)
Assembly dynamics of supramolecular
protein-DNA complexes studied by single-molecule
fluorescence microscopy
Proefschrift
ter verkrijging van de graad van doctor aan de
Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
maandag 20 februari 2017 om 09.00 uur
door
Sarah Annette Stratmann
geboren op 6 april 1986
Promotor
Prof. dr. A.M. van Oijen
Beoordelingscommissie
Prof. dr. G. Maglia
Prof. dr. S. Diez
Contents
Chapter 1: Introduction 9
1.1 DNA metabolism 10 1.2 DNA replication 10 1.3 DNA sensing by the auto-immune system 12 1.4 Aim of this thesis 14 1.5 References 15
Chapter 2: DNA replication at the single-molecule level 19
2.1 Introduction 20 2.2 Experimental strategies to image single molecules 22 2.2.1 Getting proteins to shine 22 2.2.2 Trapping and pulling at individual DNA molecules 27 2.3 Replication machineries 28 2.3.1 Model systems for single-molecule studies 30 2.3.2 Replication-fork assembly pathways 34 2.3.3 Leading and lagging-strand coordination 36 2.3.4 Polymerase dynamics 38 2.3.5 In vivo studies on the E. coli replisome 40 2.3.6 Replication in the context of eukaryotic cell division 44 2.4 Conclusions and Outlook 48 2.5 References 48
Chapter 3: Single-molecule studies of DnaB loading and dynamics at the Escherichia coli
replication fork 59
3.1 Introduction 60
3.2 Results 62
3.2.1 DnaB – association kinetics at forked DNA 62 3.2.2 Loading of multiple helicases at the replication fork 64 3.2.3 DnaB is stably integrated into the replisome during unwinding 65
3.4 Materials and Methods 68 3.5 Supplementary data 70
3.6 References 74
Chapter 4: The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by
scanning along the duplex 77
4.1 Introduction 78 4.2 Results and Discussion 78 4.3 Materials and Methods 84 4.4 Supplementary data 88 4.5 References 92
Chapter 5: Bisecting microfluidic channels with metallic nanowires fabricated by nano-skiving: Applications in flow sensing and single-molecule fluorescence studies 95
5.1 Introduction 96 5.2 Results and Discussion 97 5.2.1 Fabrication 97 5.2.2 Hot-wire anemometry 99 5.2.3 Suspended DNA curtains 101 5.2.4 Single-molecule studies on suspended DNA curtains 104 5.3 Conclusion 106 5.4 Materials and Methods 107 5.6 References 113
Chapter 6: Summary - Samenvatting 115
Summary and future perspectives 116 Samenvatting en toekomstperspectief 118
References 121
