DNA-Protein Interactions

Methods in Molecular Biology John M, Walker, SERIES EDITOR

37. In Vitro Transcription and Translation Protocols, edited by Martin J. Tymma, 1995

36. Peptide Analysis Protocols, edited by Michael W. Pennington and Ben M. Dunn, 1994

35. Peptide Synthesis Protocols, edited by Ben M. Dunn and Michael W. Pennington, 1994

34. Tmnnmocytochemical Methods and Protocols, edited by Lorette C. Javoia, 1994

33. In Situ Hybridization Protocols, edited by JL H. Andy Choo, 1994 32. Basic Protein and Peptide Protocols, edited by John M. Walker, 1994 31. Protocols for Gene Analysis, edited by Adrian J. Harwood, 1994 30. DNA-Protein Interactions, edited by G. Geoff Kneale, 1994 29. Chromosome Analysis Protocols, edited by John R. Goaden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by

Peter G. laaac, 1994 27. Biomembrane Protocols: //. Architecture and Function, edited by

John M. Graham and Joan A. Higgina, 1994 26. Protocols for Oligonucleotide Coi^ugates, edited by Sudhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited hy Hugh G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Techniques,

edited by Chriatopher Jones, Barbara Mulloy, and Adrian H. Thomaa, 1994

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993 20. Protocols for Oligonucleotides and Analogs, edited by

Sudhir Agrawal, 1993 19. Biomembrane Protocols: /. Isolation and Analysis, edited by

John M. Graham and Joan A. Higgina, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Chriatopher Jonea,

Barbara Mulloy, and Adrian H. Thomaa, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCB Protocols, edited by Bruce A. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F, Hounaell,

1993 13. Protocols in Molecular Neurobiology, edited by Alan Longataff

and Patricia Reveat, 1992 12. Pulsed-Field Gel Electrophoresis, edited by Margit Burmeiater

and Levy Ulanovaky, 1992 11. Practical Protein Chromatography, edited by Andrew Kenney

and Suaan Fowell, 1992 10. Immunochemical Protocols, edited by Margaret M. Manaon, 1992

Earlier volumes are still available. Contact Humana for detaila.

Methods in Molecular Biology

DNA-Protein Interactions Principles and Protocols

Edited by

G. Geoff Kneale School of Biological Sciences; University of

Portsmouth, Portsmouth, UK

H u m a n a P r e s s ^ j ^ Totowa, New Jersey

© 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Main entry under title:

Methods in molecular biology.

DNA-protein interactions: principles and protocols / edited by G. Geoff Kneale. p. cm.—(Methods in molecular biology; 30)

Includes index. ISBN 0-89603-256-6 1. DNA-protein interactions. I. Kneale, G. Geoff. II. Series:

Methods in molecular biology (Totowa, N.J.); 30. QP624.75.P74D57 1994 574.87'3282—dc20 93-21448

CIP

Preface

The study of protein-nucleic acid interactions is currently one of the most rapidly growing areas of molecular biology. DNA binding proteins are at the very heart of the regulation and control of gene expression, replication, and recombination: Enzymes that recognize and either modify or cleave specific DNA sequences are equally important to the cell. Some of the techniques reported in this volume can be used to identify previously unknown DNA binding proteins from crude cell extracts. Virtually all are capable of giving direct information on the molecular basis of the interaction—the location of the DNA binding site; the strength and specificity of binding; the identities of individual groups on specific bases involved in binding; the specific amino acid residues of the protein that interact with the DNA; or the effects of protein binding on gross conformation and local structure of DNA.

The recognition of DNA sequences by proteins is a complex phenomenon, involving specific hydrogen bonding contacts to the DNA bases ("direct readout") and/or interactions with the sugar-phos­phate backbone ("indirect readout"). The latter interactions can also be highly specific because of sequence-dependent conformational changes in the DNA. In addition, intercalation of planar aromatic amino acid side-chains between the DNA bases can occur, most notably with single-stranded DNA binding proteins. Furthermore, when bound, many DNA binding proteins induce drastic structural changes in the DNA as an integral part of their function. The advan­tages of using complementary techniques to investigate the complexi­ties of protein-DNA interactions will be clear, since each has advantages and disadvantages for specific tasks.

The early chapters in this volume cover DNA footprinting and related techniques, including "protection" methods (which probe accessibility of a DNA sequence to a reagent) and "interference" tech-

vi Preface

niques (which investigate the effects on DNA binding of modifica­tion of groups in the DNA recognition sequence). The results, even with the same reagent, are rarely identical, since the former picks up contacts that may not be essential for DNA binding. Ideally, both should be attempted. Although the "interference" techniques cannot be used in vivo, they are often more sensitive than "protection" techniques when applied to crude extracts. The variation between probes in sequence selectivity, site of reaction, and conformational dependence gives rise to the wide range of related techniques now in use.

Chapter 1 deals with DNase I footprinting and, like the Exoin tech­nique described in Chapter 2, is a relatively mild procedure that is most likely to identify the intact DNA binding site. However, because of the bulky size of the enzymatic probes, the use of chemical probes is recom­mended for higher resolution studies. Hydroxyl radical cleavage (Chap­ters 3 and 4) is relatively sequence-independent and attacks the DNA backbone. It can be used as either a protection or an interference method. Copper-phenanthroline footprinting (Chapter 5) is also relatively nonspecific regarding sequence since it cleaves the deoxyribose ring, probably in the minor groove. Dimethyl sulfate is another reagent that can be used for both protection and interference experiments (Chapter 6); it is highly specific for purines and can distinguish minor and major groove contacts, but favors N7 of guanine in the major groove.

Diethyl pyrocarbonate (Chapter 7) is a probe for protein-induced conformational changes in the DNA, being particularly useful in the analysis of Z-DNA formation; it preferentially attacks guanine bases. Osmium tetroxide, on the other hand, attacks thymines and has proved useful in the identification of cruciform-like structures (Chapter 8). Singlet oxygen provides a probe of structural deformation in DNA; it is not appreciably sequence selective in double helical DNA (Chap­ter 9). Both the ethylation interference technique (Chapter 10) and uranyl photofootprinting (Chapter 11) are available for the analysis of contacts with the phosphate groups of the DNA backbone.

The following six chapters deal with methods to investigate the protein component of a nucleoprotein complex. Chapters 12 and 13 describe chemical modification and proteolysis of nucleoprotein com­plexes, techniques that could be considered the analogs of chemical and enzymatic DNA footprinting techniques. The latter approach is

Preface vii

useful for the investigation of DNA binding domains within a pro­tein. The following two chapters cover methods for the overexpression of DNA binding proteins and their domains; both take advantage of fusion techniques. Chapter 14 describes a general strategy (making use of PCR) for large-scale expression of putative DNA binding domains that can be used in subsequent in vitro studies. Chapter 15 describes an alternative method using GST fusions, and its application to the overexpression of eukaryotic transcription factors. If the gene for a DNA binding protein has been cloned, then site-directed mutagenesis pro­vides an invaluable tool for the dissection of structure-function rela­tionships; two different approaches are reported (Chapters 16 and 17). The latter chapter includes the use of saturation mutagenesis for the analysis of functional requirements for a given amino acid resi­due at a particular site in the protein.

Crosslinking techniques for the investigation of protein-DNA interactions are presented in the following two chapters. Such meth­ods are valuable in identifying specific DNA sequences that interact with a given protein, and can sometimes be used where other techniques fail. Indeed, subsequent biochemical analysis of the protein may also reveal specific amino acid residues in close proximity to the DNA binding site. A potential problem with all such methods is that the yield of crosslinked product is often low. In Chapter 18, this is over­come by use of UV laser irradiation. Chapter 19 describes the use of photoaffinity labeling, where the modified base 8-azidoadenine is introduced into the target DNA sequence.

Quantitative determination of DNA binding affinities can be estab­lished by a number of techniques. The filter binding assay remains a useful method for many purposes, and is described in Chapter 20. However, the gel-shift assay (Chapter 21) is of more general utility and can often allow the resolution of multiple species of DNA-protein complexes. Indeed, the gel-shift technique forms a part of many of the methods described in earlier chapters. A specific application of the gel-shift assay is for the analysis of protein-induced DNA bend­ing, for which specialized vectors have been developed (Chapter 22). It also forms part of the binding site-selection technique covered in Chapter 23, one of a number of recently developed protocols for the determination of specific base requirements in a DNA binding sequence.

via Preface

This protocol employs solid-phase chemical sequencing for the analy­sis of preferred binding sites.

Spectroscopic techniques are especially useful in the quantita­tive study of protein-DNA interactions when highly purified compo­nents are available. Fluorescence methods can give information on stoichiometry, binding constants, and frequently also on the interac­tion of aromatic amino acid residues with the DNA (Chapter 24). In certain cases, however, the intrinsic fluorescence of the protein can­not be used to monitor DNA binding. Chapter 25 describes an alter­native method based on the displacement of the fluorescent probe ANS from the DNA binding site. Circular dichroism can monitor con­formational changes in either the protein or the DNA component of a complex (Chapter 26). Again, it is often the combination of such tech­niques that provides particularly valuable insights into the nature of the interaction. For more direct structural information, especially for large nucleoprotein complexes, electron microscopy can provide a low resolution image (Chapter 27). Ultimately, X-ray crystallogra­phy is the preferred technique for high resolution structural data and Chapter 28 provides a useful source of information for getting started in this direction. Clearly, a detailed description of X-ray diffraction techniques is well beyond the scope of this volume. The same is true of NMR spectroscopy, which is also proving instrumental in our under­standing of protein-nucleic acid interactions.

The remaining chapters in this volume describe functional assays for a variety of proteins that interact with DNA. Such studies are vital to complement the structural techniques, but are of necessity more specific. Chapters 29 and 30 deal with protocols for the assay of restriction enzyme activity using synthetic oligonucleotides and plasmids, respectively, as substrates. Chapter 31 describes a number of techniques for the assay of transcription factor activity, and Chap­ter 32 describes an assay for proteins involved in genetic recombination.

The aim in describing these techniques was to provide complete protocols wherever possible, so that they could be used by a relative newcomer to the field. Inevitably, this involves some duplication between chapters since there is some overlap of methodologies; none­theless, this has been kept to a minimum. The introduction to each chapter provides an overview of the method, a brief theoretical back-

Preface ix

ground and, when relevant, a discussion of its advantages and draw­backs. In keeping with the format of other volumes in the series, there is a Materials section in each chapter listing all the reagents that will be required for the procedures. The individual procedures vary in length and complexity from chapter to chapter, but in each case they are broken down into a series of small steps. Finally, the Notes sec­tion provides hints or further explanation of the rationale underlying the technique, which should prove invaluable to the experimenter.

The reader should know that it was impossible to cover every variation on a given technique in one volume. Inevitably there will be omissions. However, the editor believes that the methods included will provide a sufficiently broad range that the experimenter will be able to tackle problems in every area of protein-nucleic acid interac­tions from a number of different perspectives, and so build up a more complete understanding of the molecular processes involved.

G. Geoff Kneale

Contents

Preface v Contributors xv CH. 1. DNase I Footprinting,

Benott Leblanc and Tom Moss / CH. 2. Footprinting with Exonuclease III,

Willi Metzger and Hermann Neumann 11 CH. 3. Hydroxyl Radical Footprinting,

Peter Schickor and Hermann Heumann 21 CH. 4. Hydroxyl Radical Interference,

Peter Schickor and Hermann Heumann 33 CH. 5. 1,10-Phenanthroline-Copper Ion Nuclease Footprinting of DNA-

Protein Complexes in Situ Following Mobility-Shift Electrophoresis Assays,

Athanasios G. Papavassilou 43 CH. 6. Identification of Protein-DNA Contacts with Dimethyl Sulfate:

Methylation Protection and Methylation Interference, Peter E. Shaw and A. Francis Stewart 79

CH. 7. Diethyl Pyrocarbonate as a Probe of Protein-DNA Interactions, MichaelJ. McLean 89

CH. 8. Osmium Tetroxide Modification and the Study of DNA-Protein Interactions,

James A. McClellan 97 CH. 9. Diffusible Singlet Oxygen as a Probe of DNA Deformation,

Malcolm Buckle and Andrew A. Travers 113 CH. 10. Ethylation Interference,

Iain Manfield and Peter G. Stockley 125 CH. 11. Uranyl Photofootprinting of DNA-Protein Complexes,

Peter E. Nielsen 141 CH. 12. Nitration of Tyrosine Residues in Protein-Nucleic Acid Complexes,

Simon E. Plyte 151 CH. 13. Limited Proteolysis of Protein-Nucleic Acid Complexes,

Simon E. Plyte and G. Geoff Kneale 161 CH. 14. Cloning and Expression of DNA Binding Domains Using PCR,

Daniel G. Fox and G. Geoff Kneale 169 X

Contents xi

CH. 15. Overexpression and Purification of Eukaryotic Transcription Factors as Glutathione-S-Transferase Fusions in E. coli,

Kevin G. Ford, Alan J. Whitmarsh, and David P. Hornby 185 CH. 16. Site-Directed Mutagenesis by the Cassette Method,

Andrew F. Worrell 199 CH. 17. Site-Directed and Site-Saturation Mutagenesis Using Oligonucleotide

Primers, MichaelJ. O'Donohueand G. Geoff Kneale 211

CH. 18. UV Laser-Induced Protein-DNA Crosslinking, Stefan I. Dimitrov and Tom Moss 227

CH. 19. Ultraviolet Crosslinking of DNA-Protein Complexes via 8-Azidoadenine,

Rainer Meffert, Klaus Dose, Gabriele Rathgeber, and Hans-Jochen Schdfer 237

CH. 20. Filter-Binding Assays, Peter G. Stockley 251

CH. 21. The Gel Shift Assay for the Analysis of DNA-Protein Interactions, John D. Taylor, Alison J. Ackroyd, and Stephen E. Halford ...263

CH. 22. Improved Plasmid Vectors for the Analysis of Protein-Induced DNA Bending,

Christian Zwieb and SankarAdhya 281 CH. 23. Determination of Sequence Preferences of DNA Binding Proteins

Using Pooled Solid-Phase Sequencing of Low Degeneracy Oligonucleotide Mixtures,

Joseph A. Gogos and FOtis C. Kafatos 295 CH. 24. Analysis of DNA-Protein Interactions by Intrinsic Fluorescence,

Mark L. Carpenter and G. Geoff Kneale 313 CH. 25. A Competition Assay for DNA Binding Using the Fluorescent Probe

ANS, Ian Taylor and G. Geoff Kneale 327

CH. 26. Circular Dichroism for the Analysis of Protein-DNA Interactions, Mark L. Carpenter and G. Geoff Kneale 339

CH. 27. Electron Microscopy of Protein-Nucleic Acid Complexes: Uniform Spreading and Determination of Helix Handedness,

Carlo W. Gray 347 CH. 28. Reconstitution of Protein-DNA Complexes for Crystallization,

Rachel M. Conlin and Raymond S. Brown 357 CH. 29. Assay of Restriction Endonucleases Using Oligonucleotides,

Bernard A. Connolly 371 CH. 30. Assays for Restriction Endonucleases Using Plasmid Substrates,

Stephen E. Halford, John D. Taylor, Christian L. M. Vermote, and I. Barry Vipond 385

xii Contents

CH. 31. Assays for Transcription Factor Activity, Stephen Busby, Annie Kolb, and Stephen Minchin 397

CH. 32. An Assay for in Vitro Recombination Between Duplex DNA Molecules,

Berndt Muller and Stephen C. West 413

Index 425

Contributors

ALISON J. ACKROYD • Department of Biochemistry, University of Texas at Dallas, Southwestern Medical Center, Dallas, TX

SANKAR ADHYA • Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD

RAYMOND S. BROWN • Howard Hughes Medical Institute, Harvard

University, Boston, MA MALCOLM BUCKLE • Institute Pasteur, Paris, France STEPHEN BUSBY • School of Biochemistry, University of Birmingham,

Birmingham, UK MARK L. CARPENTER • Sir William Dunn School of Pathology,

University of Oxford, Oxford, UK RACHEL M . CONLIN • Howard Hughes Medical Institute, Harvard

University, Cambridge, MA BERNARD A. CONNOLLY • Department of Biochemistry and Genetics,

The University, Newcastle-upon-Tyne, UK STEFAN I. DIMITROV • Centre de Recherche en Cancerologie de

I'Universite Laval, Quebec, Canada KLAUS DOSE • Institiit fiir Biochemie derJohannes Gutenberg-

Universitdt, Mainz, Germany KEVIN G. FORD • Krebs Institute, Department of Molecular Biology

and Biotechnology, University of Sheffield, Sheffield, UK DANIEL G. FOX • Biophysics Laboratories, School of Biological Sciences,

University of Portsmouth, Portsmouth, UK JOSEPH A. Gooos • Department of Cellular and Developmental

Biology, The Biological Laboratories, Harvard University, Cambridge, MA

CARLA W . GRAY • Program in Molecular and Cell Biology (F031), The University of Texas at Dallas, Dallas, TX

xiu

xiv Contributors

STEPHEN E . HALFORD • Department of Biochemistry, Centre for Molecular Recognition, University of Bristol, Bristol, UK

HERMANN HEUMANN • Max Planck Institute of Biochemistry, Martinsried, Germany

DAVID P. HORNBY • Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

FoTis C. KAFATOS • Institute of Molecular Biology and Biotechnology, Research Center of Crete, Crete, Greece; and Department of Cellular and Developmental Biology, The Biological Laborato­ries, Harvard University, Cambridge, MA

G. GEOFF KNEALE • Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK

ANNIE KOLB • Department de Biologie Moleculaire, Institut Pasteur, Paris, France

BENOIT LEBLANC • Centre de Recherche en Cancerologie de I'Universite Laval, Quebec, Canada

IAIN MANFIELD • Department of Genetics, University of Leeds, Leeds, UK JAMES A. MCCLELLAN • Biophysics Laboratories, School of Biological

Sciences, University of Portsmouth, Portsmouth, UK MICHAEL J. MCLEAN • Cambridge Research Biochemicals, Cheshire, UK RAINER MEFFERT • Institiit fiir Biochemie derfohannes Gutenberg-

Universitdt, Mainz, Germany WILLI METZGER • Max Planck Institute of Biochemistry, Martinsried,

Germany STEPHEN MINCHIN • School of Biochemistry, University of Birmingham,

Birmingham, UK TOM MOSS • Centre de Recherche en Cancerologie de I'Universite

Laval, Quebec, Canada BERNDT MULLER • Imperial Cancer Research Fund, Clare Hall

Laboratories, South Mimms, UK PETER E . NIELSEN • Research Center for Medical Biotechnology,

Department of Biochemistry, The Panum Institute, Copenhagen, Denmark

MICHAEL J. O'DONOHUE • Laboratory of Organic Chemistry, Univer­sity of Paris V, Paris, France

Contributors xv

ATHANASIOS G. PAPAVASSILIOU • European Molecular Biology Labo­ratory, Heidelberg, Germany

SIMON E . PLYTE • Ludwig Institute for Cancer Research, London, UK GABRIELE RATHGEBER • Institiitfur Biochemie der Johannes

Gutenberg-Universitdt, Mainz, Germany HANS-JOCHEN SCHAFER • Institiit fiir Biochemie der Johannes

Gutenberg-Universitdt, Mainz, Germany PETER SCHICKOR • Max Planck Institute of Biochemistry,

Martinsried, Germany PETER E. SHAW • Max Planck Institiit fiir Immunbiologie, Freiburg -

Zdhringen, Germany A. FRANCIS STEWART • European Molecular Biology Laboratories,

Heidelberg, Germany PETER G. STOCKLEY • Department of Genetics, University of Leeds,

Leeds, UK IAN TAYLOR • Biophysics Laboratories, School of Biological Sciences,

University of Portsmouth, Portsmouth, UK JOHN D . TAYLOR • Department of Biochemistry, Duke University

Medical Center, Durham, NC ANDREW A. TRAVERS • MRC Laboratory of Molecular Biology,

Cambridge, UK CHRISTIAN L . M . VERMOTE • Department of Biochemistry, Centre

for Molecular Recognition, University of Bristol, Bristol, UK I. BARRY ViPOND • Department of Biochemistry, Centre for Molecular

Recognition, University of Bristol, Bristol, UK STEPHEN C. WEST • Imperial Cancer Research Fund, Clare Hall

Laboratories, South Mimms, UK ALAN J. WHITMARSH • Krebs Institute, Department of Molecular

Biology and Biotechnology, University of Sheffield, Sheffield, UK ANDREW F. WORRALL • Department of Biochemistry, University

of Southampton, Southampton, UK CHRISTIAN ZWIEB • Department of Molecular Biology, The University

of Texas Health Center at Tyler, Tyler, TX