DNA-Protein Interactions A Practical Approach

by Travers, Andrew; Buckle, Malcolm

• ISBN13: 9780199636921
• ISBN10: 0199636923
• Edition: 1st
• Format: Hardcover
• Copyright: 2000-10-12
• Publisher: Oxford University Press

Summary

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.

Table of Contents

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 DNA	12	(2)
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 with DNA	27	(1)
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 site	48	(1)
Generating of the monomer fragment	48	(5)
Monomer oligomerization and cloning	53	(2)
Strategies for further improvement in the process of plasmid construction	55	(1)
Binding the protein and generating the ΔL	56	(1)
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 binding to differently positioned nucleosomes	68	(2)
Fractionation of protein-DNA complexes prior to further analysis	70	(1)
Estimation of dissociation constants	70	(4)
Some other applications for the bandshift assay	74	(3)
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 energy transfer	78	(3)
Orientation effects of the dyes	79	(1)
Influence on the spectroscopic properties of the dyes by other mechanisms than FRET	80	(1)
The influence of donor-acceptor distance distributions	81	(1)
Experimental determination of FRET efficiencies	81	(4)
Normalizing the enhanced steady-state fluorescence of the energy acceptor	82	(2)
Measuring changes in the fluorescence anisotropy of the energy donor	84	(1)
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 RNA structures	86	(1)
Structural interpretation of FRET efficiencies	87	(8)
Modelling the dye positions at the DNA helix ends	88	(2)
Modelling nucleic acid conformations and protein-DNA interactions	90	(2)
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 systems	97	(5)
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 complexes	115	(2)
Image analysis: DNA contour length measurements	117	(1)
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 experiment	121	(1)
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 quantities	127	(1)
Heat capacity	128	(1)
Linked functions: ion binding and protonation changes	129	(1)
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 complexes	136	(1)
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 crosslinking	143	(8)
Crosslinking of RNA polymerase-promoter complexes	143	(3)
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 nucleoside assay	163	(1)
Quantification and interpretation of hydroxyl radical footprinting data	163	(2)
Experimental procedures	165	(10)
Preparation of the end-labelled DNA	165	(4)
Formation of the DNA-protein complexes and attack by the hydroxyl radicals	169	(2)
Interference studies: the missing nucleoside assay	171	(1)
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. Protection factor	178	(3)
Principle of the method	178	(2)
Protection of plasmids by a DNA-binding protein	180	(1)
Determination of breakage sites using DNA restriction fragments. Protected sites: footprints	181	(6)
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 signals in vitro	203	(7)
Samples for in vivo footprinting	210	(1)
Measurement of in vivo binding under different growth conditions	211	(2)
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 footprinting	230	(1)
Limitations of the procedures	230	(1)
Practical applications	231	(8)
Crosslinking of histones to chromatosome and core nucleosome DNA	231	(1)
Crosslinking and mapping of crosslinking sites on DNA	232	(2)
Mapping histone positions using EDTA conjugates	234	(3)
References	237	(2)
Kinetic analysis of enzyme template interactions. Nucleotide incorporation by DNA dependent RNA and DNA polymerases
Bianca Sclavi
Pascal Roux
Introduction	239	(1)
Purification of DNA fragments	240	(1)
Abortive initiation assays to probe interactions between RNA polymerases and prokaryotic promoters	241	(8)
Principle of the assay (3,4)	241	(1)
Steady-state assays: radioactive incorporation of an α-32 P-labelled NTP into the abortive product	242	(3)
Analysis of the enzymatic reaction	245	(1)
Lag assay. Quantitative characterization of the `on' process	246	(1)
Residence time of RNAP at the promoter. Dissociation rate constants	247	(1)
The fluorescent assay	248	(1)
Changes in mechanism for open complex formation and/or escape	248	(1)
Elongation assays to study the interactions between DNA polymerases and their templates	249	(8)
Introduction	249	(1)
Principle of the assay	250	(4)
Sequential incorporation of several dNTP during a run-off assay	254	(1)
Acknowledgements	255	(1)
References	255	(2)
Kinetics of DNA interactions surface plasmon resonance Spectroscopy
Bjorn Persson
Malcolm Buckle
Peter G. Stockley
Principles of surface plasmon resonance technology	257	(5)
Introduction	257	(1)
Principle of detection	257	(1)
Surface chemistry	258	(1)
Liquid handling system	259	(1)
Data handling	260	(2)
SPR-assays of protein-DNA interactions	262	(5)
Experimental design	262	(1)
DNA immobilization	263	(1)
Met J sensorgrams	264	(2)
Kinetics	266	(1)
The sensor chip surface	266	(1)
SPR assays of polymerase action	267	(6)
Kinetics of RNA polymerase interactions with immobilized DNA	267	(1)
DNA immobilization	268	(3)
RNA polymerase binding to immobilized DNA	271	(2)
DNA hybridization	273	(8)
DNA immobilization	275	(1)
Hybridization analysis	276	(2)
Effects of mismatches	278	(1)
Sequence screening	278	(1)
Acknowledgements	279	(1)
References	279	(2)
Quantitative DNase I kinetics footprinting
A. K. M. M. Mollah
Michael Brenowitz
Introduction	281	(1)
Quench-flow DNase I footprinting	282	(9)
Preliminary considerations	282	(1)
Reagent preparation	283	(1)
Conducting a quench-flow DNase I `footprinting' experiment	284	(2)
Experimental conditions and selection of data points to be acquired	286	(1)
Manual mixing DNase I kinetics footprinting	286	(1)
Data reduction and analysis	287	(3)
Accuracy and precision of the measurements	290	(1)
References	290	(1)
Analysis of DNA-protein interactions by time-resolved fluorescence spectroscopy
E. H. Z. Thompson
D. P. Millar
Introduction	291	(1)
Time-resolved fluorescence techniques	291	(7)
Fluorescence lifetime decays	292	(2)
Fluorescence anisotropy decay	294	(4)
Experimental guidelines	298	(3)
Guidelines for fluorophore choice	298	(1)
DNA labelling	299	(1)
Instrumentation	300	(1)
Examples	301	(6)
TyrR-DNA interaction: a simple example of fluorescence lifetime and anisotropy parameters	301	(2)
Klenow fragment-DNA interaction: a complex example of time-resolved anisotropy	303	(3)
References	306	(1)
Analysis of protein-DNA interactions in complex nucleoprotein assemblies
Iain K. Pemberton
Introduction	307	(1)
Protocol for the transfer of a labelled nucleotide to a protein subunit by UV irradiation	307	(8)
Radiolabelling of the DNA fragment at a specific nucleotide position	308	(5)
Identification of subunit interactions by UV irradiation and nuclease digestion	313	(2)
Specific example of promoter recognition by the E. coli RNA polymerase	315	(2)
Perspectives	317	(2)
References	317	(2)
Site-specific protein-DNA photocrosslinking
Tae-Kyung Kim
Thierry Lagrange
Danny Reinberg
Introduction	319	(1)
Procedure	319	(14)
Outline of procedure	319	(2)
Preparation of site-specifically derivatized DNA fragment	321	(6)
Photocrosslinking	327	(6)
Representative data	333	(1)
Prospects	334	(3)
Acknowledgements	334	(1)
References	334	(3)
DNA-protein complexes analysed by electron microscopy and cryo-microscopy
Eric Le Cam
Etienne Delain
Eric Larquet
Francoise Culard
Jean A.H. Cognet
Introduction	337	(1)
Electron microscopy of DNA-protein complexes spread on carbon film support	338	(7)
Methods used to observe nucleic acids and nucleoprotein complexes	338	(4)
Characterization and method for the analysis of the binding of protein MC1 to DNA. Measurement of DNA curvature and flexibility	342	(3)
Cryo-electron microscopy	345	(6)
Methods used to observe nucleic acids and nucleoprotein complexes	345	(2)
3D-reconstruction of DNA molecules	347	(2)
Acknowledgements	349	(1)
References	350	(1)
Characterization of T7 RNA polymerase protein-DNA interactions during the initiation and elongation phases
Dmitry Temiakov
Pamela E. Karasavas
William T. McAllister
Introduction	351	(2)
Purification of T7 RNA polymerase	353	(2)
Formation of halted complexes in a conserved sequence context	355	(1)
Walking of T7 RNAP on a DNA template	356	(3)
Purification of crosslinked protein	359	(1)
Mapping of crosslink sites of nucleic acids to T7 RNA polymerase by protease digestion	360	(2)
Purification of N-terminal fragments of T7 RNAP	362	(3)
References	363	(2)
A1 List of suppliers	365	(6)
Index	371