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Preface v
List of protocols xvii
Abbreviations xxi
Expression systems
Reinhard Grisshammer
Christian Kambach
Christopher G. Tate
Introduction 1 (1)
Expression in Escherichia coli 1 (10)
Target genes and cDNAs 2 (1)
Codon usage 2 (1)
Fusion proteins 3 (1)
Prokaryotic expression vectors 3 (3)
E. coli hosts 6 (1)
Growth conditions 6 (1)
Co-expression of proteins 7 (2)
Troubleshooting 9 (2)
The baculovirus expression system 11 (14)
Introduction 11 (1)
Choice of transfer vector and baculovirus 12 (2)
DNA
Choice of host cells and growth conditions 14 (2)
Constructing a recombinant baculovirus 16 (6)
Optimization of expression 22 (1)
Acknowledgements 23 (1)
References 23 (2)
Gel electrophoresis and bending in
cisplatin-modified linear DNA
Jean-Marc Malinge
Annie Schwartz
Marc Leng
Introduction 25 (1)
Gel electrophoresis 25 (2)
Background on gel electrophoresis 25 (1)
Experimental procedure 26 (1)
Chemistry of cisplatin and transplatin 27 (4)
Reactivity of cisplatin and transplatin 27 (1)
with DNA
Platination of the oligonucleotides 28 (2)
Analysis of the adducts 30 (1)
Synthesis of the multimers 30 (1)
Analysis of the results 31 (6)
References 34 (3)
Use of DNA microcircles in protein-DNA
binding studies
Dominique Payet
Introduction 37 (1)
DNA circularization 37 (4)
Oligonucleotide purification 38 (2)
Oligonucleotide design 40 (1)
Examples 41 (6)
DNA circularization by HMG-D 41 (1)
HMG-D circle interaction 42 (2)
References 44 (3)
Use of topology to measure protein-induced
DNA bend and unwinding angles
Leonard C. Lutter
Christopher E. Drabik
Herbert R. Halvorson
Introduction 47 (1)
Plasmid construction 48 (8)
Designing the plasmid 48 (1)
Initial cloning of the protein-binding 48 (1)
site
Generating of the monomer fragment 48 (5)
Monomer oligomerization and cloning 53 (2)
Strategies for further improvement in the 55 (1)
process of plasmid construction
Binding the protein and generating the 56 (1)
ΔL
Site saturation 56 (1)
Non-specific binding 57 (1)
Gel electrophoresis 57 (1)
Data processing 57 (8)
Capturing the data 58 (3)
Taming the data 61 (3)
Acknowledgement 64 (1)
References 64 (1)
Bandshift, gel retardation or electrophoretic
mobility shift assays
Louise Fairall
Memmo Buttinelli
Gianna Panetta
Introduction 65 (1)
Some general considerations 65 (1)
Gel systems 66 (2)
Electrophoresis buffers 66 (1)
Sample buffers 67 (1)
Agarose gels 67 (1)
Polyacrylamide gels 67 (1)
Visualization of gels 68 (1)
Uses of the bandshift assay 68 (9)
Fractionation of transcription factor 68 (2)
binding to differently positioned
nucleosomes
Fractionation of protein-DNA complexes 70 (1)
prior to further analysis
Estimation of dissociation constants 70 (4)
Some other applications for the bandshift 74 (3)
assay
Practical aspects of fluorescence resonance
energy transfer (FRET) and its applications
in nucleic acid biochemistry
Frank Stuhmeier
Robert M. Clegg
Alexander Hillisch
Stephan Diekmann
Introduction 77 (1)
Basic principles of fluorescence resonance 78 (3)
energy transfer
Orientation effects of the dyes 79 (1)
Influence on the spectroscopic properties 80 (1)
of the dyes by other mechanisms than FRET
The influence of donor-acceptor distance 81 (1)
distributions
Experimental determination of FRET 81 (4)
efficiencies
Normalizing the enhanced steady-state 82 (2)
fluorescence of the energy acceptor
Measuring changes in the fluorescence 84 (1)
anisotropy of the energy donor
Preparation of dye-labelled DNA structures 85 (1)
Labelling with an amino-reactive compounds 85 (1)
Purification by denaturing and native PAGE 86 (1)
Characterization of dye-labelled DNA and 86 (1)
RNA structures
Structural interpretation of FRET 87 (8)
efficiencies
Modelling the dye positions at the DNA 88 (2)
helix ends
Modelling nucleic acid conformations and 90 (2)
protein-DNA interactions
References 92 (3)
Determination of DNA-ligand interactions by
fluorescence correlation spectroscopy
J. Langowski
M. Tewes
Introduction 95 (2)
Theoretical foundation of FCS 97 (9)
Concentration fluctuations in small 97 (5)
systems
Construction of a typical FCS instrument 102 (4)
Sample requirements 106 (1)
Some examples from current research 106 (7)
Triplex formation 106 (1)
NtrC protein 107 (2)
Vimentin oligomerization 109 (1)
Acknowledgement 110 (1)
References 110 (3)
DNA wrapping in Escherichia coli RNA
polymerase open promoter complexes revealed
by scanning force microscopy
Claudio Rivetti
Martin Guthold
Carlos Bustamante
Introduction 113 (1)
Scanning force microscopy 114 (3)
Imaging protein-DNA complexes with the SFM 114 (1)
Preparation and deposition of RNAP-DNA 115 (2)
complexes
Image analysis: DNA contour length 117 (1)
measurements
DNA contour length analysis 117 (4)
DNA alone 117 (1)
Open promoter complexes 118 (2)
Analysis of the DNA arms 120 (1)
Heparin-resistant complexes as a control 121 (1)
experiment
Concluding remarks 121 (4)
Acknowledgements 123 (1)
References 123 (2)
Microcalorimetry of protein-DNA interactions
Alan Cooper
Introduction 125 (1)
Thermodynamics: all you need to know 126 (4)
Basics 126 (1)
Equilibrium and `standard' thermodynamic 127 (1)
quantities
Heat capacity 128 (1)
Linked functions: ion binding and 129 (1)
protonation changes
Microcalorimetry instrumentation and methods 130 (4)
Examples 134 (7)
DSC of DNA-repressor interactions 134 (1)
ITC of DNA-repressor interactions 135 (1)
DSC of protein-single-stranded DNA 136 (1)
complexes
ITC of protein-RNA interactions 137 (1)
References 138 (3)
Protein-DNA crosslinking with formaldehyde in
vitro
Konstantin Brodolin
Introduction 141 (1)
Chemistry of formaldehyde crosslinking 141 (2)
Practical application of formaldehyde 143 (8)
crosslinking
Crosslinking of RNA polymerase-promoter 143 (3)
complexes
Identification of crosslinked species 146 (2)
Mapping of crosslinking sites on DNA 148 (1)
References 149 (2)
Solid-phase DNAse I footprinting
Raphael Sandaltzopoulos
Peter B. Becker
Introduction 151 (1)
Solid-phase footprinting protocol 151 (6)
Using a radioactive probe 151 (5)
Using a non-radioactive labelled probe 156 (1)
Applications of solid-phase footprinting 157 (4)
Acknowledgements 158 (1)
References 158 (3)
Hydroxyl radical footprinting
Annie Kolb
Tamara Belyaera
Nigel Savery
Introduction 161 (1)
Principle of the procedure 162 (3)
Chemistry of the reaction 162 (1)
Principle of hydroxyl radical footprinting 162 (1)
Interference method: the missing 163 (1)
nucleoside assay
Quantification and interpretation of 163 (2)
hydroxyl radical footprinting data
Experimental procedures 165 (10)
Preparation of the end-labelled DNA 165 (4)
Formation of the DNA-protein complexes 169 (2)
and attack by the hydroxyl radicals
Interference studies: the missing 171 (1)
nucleoside assay
Data collection and analysis 172 (1)
References 173 (2)
Radiolytic cleavage of DNA. Mapping of the
protein interaction sites
Michel Charlier
Melanie Spotheim-Maurizot
Introduction 175 (1)
Irradiation of samples 176 (2)
Determination of FSB yield using plasmids. 178 (3)
Protection factor
Principle of the method 178 (2)
Protection of plasmids by a DNA-binding 180 (1)
protein
Determination of breakage sites using DNA 181 (6)
restriction fragments. Protected sites:
footprints
Principle of the method 181 (3)
Radiolysis of naked DNA 184 (1)
Footprint of a protein on DNA 184 (3)
Conclusion 187 (2)
References 187 (2)
UV-laser photoreactivity of nucleoprotein
complexes in vitro
Malcolm Buckle
Christophe Place
Iain K. Pemberton
Introduction 189 (1)
UV-laser configuration 189 (12)
Hardware set-up 189 (3)
Conditions for photo-irradiation 192 (1)
Identification of photoreactive species 193 (7)
References 200 (1)
In vivo UV-laser footprinting
Frederic Boccard
Sylvie Dethiollaz
Manuel Engelhorn
Johannes Geiselmann
Introduction 201 (1)
The scope of UV-laser footprinting 201 (2)
Detecting DNA-protein interactions in vivo 201 (1)
Principle of the reaction 202 (1)
Practical considerations 203 (10)
Equipment 203 (1)
Calibration of the UV-footprinting 203 (7)
signals in vitro
Samples for in vivo footprinting 210 (1)
Measurement of in vivo binding under 211 (2)
different growth conditions
Conclusions and perspectives 213 (2)
References 213 (2)
Digitization and quantitative analysis of
footprinting gels
Judith Smith
Introduction 215 (1)
Implementation 216 (3)
Digitization 216 (1)
Analysis 216 (3)
Algorithms 219 (2)
Peak profile-fitting function 219 (1)
Variability of peak width and shape 220 (1)
Area decomposition from overlapping peaks 220 (1)
Instrument and software comparison 221 (4)
Digitization 221 (1)
Computer analysis 222 (3)
Conclusion 225 (4)
References 226 (1)
Appendix 227 (2)
Mapping histone positions in chromatin by
protein-directed DNA crosslinking and cleavage
Andrew Travers
Introduction 229 (1)
Principle of the procedure 230 (1)
Chemistry of conjugate-directed 230 (1)
footprinting
Limitations of the procedures 230 (1)
Practical applications 231 (8)
Crosslinking of histones to chromatosome 231 (1)
and core nucleosome DNA
Crosslinking and mapping of crosslinking 232 (2)
sites on DNA

DNA-Protein Interactions is a novel compilation of methods for studying the interactions of proteins with DNA. It is a rapidly advancing research area in which multidisciplinary approaches are especially valuable for solving problems and obtaining a detailed understanding of the molecularregulatory interactions involved. This book covers all the major tools that are required for the study of the large macromolecular enzymatic machines that manipulate DNA, with particular emphasis on biophysical techniques applied to the analysis of transcription and its relation to chromatinstructure. Knowledge of basic techniques is assumed, although advances in fundamental fields are covered.