Moss Tom. DNA-protein interactions: principles and protocols

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25 Site-Directed DNA Photoaffinity Labeling of RNA Polymerase III

Transcription Complexes

Jim Persinger and Blaine Bartholomew ......................................... 363

26 Use of Site-Specific Protein–DNA Photocrosslinking to Analyze

the Molecular Organization of the RNA Polymerase

II Initiation Complex

François Robert and Benoît Coulombe .......................................... 383

27 UV Laser-Induced Protein–DNA Crosslinking

Stefan I. Dimitrov and Tom Moss .................................................... 395

28 Plasmid Vectors for the Analysis of Protein-Induced

DNA Bending

Christian Zwieb and Sankar Adhya................................................. 403

29 Engineering Nucleic Acid-Binding Proteins by Phage Display

Mark Isalan and Yen Choo................................................................ 417

30 Genetic Analysis of DNA–Protein Interactions Using a Reporter

Gene Assay in Yeast

David R. Setzer, Deborah B. Schulman,

and Michael J. Bumbulis .............................................................. 431

31 Assays for Transcription Factor Activity

Virgil Rhodius, Nigel Savery, Annie Kolb,

and Stephen Busby ....................................................................... 451

32 Assay of Restriction Endonucleases Using Oligonucleotides

Bernard A. Connolly, Hsiao-Hui Liu, Damian Parry,

Lisa E. Engler, Michael R. Kurpiewski,

and Linda Jen-Jacobson ............................................................. 465

33 Analysis of DNA–Protein Interactions by Intrinsic Fluorescence

Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale....... 491

34 Circular Dichroism for the Analysis of Protein–DNA Interactions

Mark L. Carpenter, Anthony W. Oliver, and G. Geoff Kneale....... 503

35 Calorimetry of Protein–DNA Complexes and Their Components

Christopher M. Read and Ilian Jelesarov ....................................... 511

36 Surface Plasmon Resonance Applied to DNA–Protein Complexes

Malcolm Buckle.................................................................................. 535

37 Reconstitution of Protein–DNA Complexes for Crystallization

Rachel M. Conlin and Raymond S. Brown ..................................... 547

38 Two-Dimensional Crystallization of Soluble Protein Complexes

Patrick Schultz, Nicolas Bischler, and Luc Lebeau ...................... 557

Contents

39 Atomic Force Microscopy of DNA and Protein–DNA Complexes

Using Functionalized Mica Substrates

Yuri L. Lyubchenko, Alexander A. Gall,

and Luda S. Shlyakhtenko ........................................................... 569

40 Electron Microscopy of Protein–Nucleic Acid Complexes:

Uniform

Spreading of Flexible Complexes, Staining with a Uniform Thin

Layer of Uranyl Acetate, and Determining Helix Handedness

Carla W. Gray ..................................................................................... 579

41 Scanning Transmission Electon Microscopy

of DNA–Protein Complexes

Joseph S. Wall and Martha N. Simon.............................................. 589

42 Determination of Nuleic Acid Recognition Sequences by SELEX

Philippe Bouvet.................................................................................. 603

43 High DNA–Protein Crosslinking Yield with Two-Wavelength

Femtosecond Laser Irradiation

Christoph Russmann, Rene Beigang, and Miguel Beato............. 611

Appendices:

Appendix I: EMSA/Gel Shift Conditions.............................................. 617

Appendix II: DNA-Modification/Cleavage Reagents........................... 619

Index ............................................................................................................ 621

xii Contents

xiii

Contributors

SANKAR ADHYA • Laboratory of Molecular Biology, National Institutes

of Health, NCI, Bethesda, MD

ARTIN ANGERS • Division de Pathologie, Department de Biologie Médicale,

Université Laval, et Unité de Recherche en Génétique Humaine

et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise,

Québec, Canada

LAINE BARTHOLOMEW • Department of Biochemistry and Molecular Biology,

School of Medicine, Southern Illinois University, Carbondale, IL

MIGUEL BEATO • Insitute für Molekularbiologie und Tumorforshung,

Philipps-Universität Marburg, Marburg, Germany

RENE BEIGANG • Fachbereich Physik, Universität Kaiserlautern, Germany

ALAIN BÉLIVEAU • Laboratory of Molecular Endocrinologie, Centre

Hopitalier Universitaire de Québec, Université Laval, Québec, Canada

NICOLAS BISCHLER • Faculté de Médicine, IGBMC, Illkirch, France

FREDERIC BOCCARD • Centre de Génétique Moléculaire, CNRS, Yvette, France

PHILIPPE BOUVET • Laboratoire de Pharmacologie et de Biologie Structurale,

CNRS, Toulouse, France

RAYMOND S. BROWN • Laboratory of Molecular Medicine, Howard Hughes

Medical Institute, Children’s Hospital, Boston, MA

MALCOLM BUCKLE • Unité Physicochimie des Macromolécules Biologiques,

Institut Pasteur, Paris, France

MICHAEL J. BUMBULIS • Department of Molecular Biology and Microbiology,

School of Medicine, Case Western Reserve University, Cleveland,

and the Department of Biology, Baldwin-Wallace College, Berea, OH

STEPHEN BUSBY • School of Biochemistry, University of Birmingham,

Birmingham, UK

MARK L. CARPENTER • University of Oxford, Oxford, UK

DAVID R. CHAFIN • Department of Biochemistry, University of Rochester,

Rochester, NY

YEN CHOO • Laboratory of Molecular Biology, Medical Research Council,

Cambridge, UK

RACHEL M. CONLIN • Laboratory of Molecular Medicine, Howard Hughes

Medical Institute, Children’s Hospital, Boston, MA

xiv Contributors

BERNARD A. CONNOLLY • Department of Biochemistry and Genetics, Medical

School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK

ENOÎT COULOMBE • Départment de Biologie, Centre de Recherche sur

les Méchanismes d’Expression Génétique, Université de Sherbrooke,

Sherbrooke, Québec, Canada

UDMILLA DENISSOVA • Max Planck Institute of Biochemistry, Martinsried, Germany

TEFAN

I. D

IMITROV

• Faculté de Médecine, Institut Albert Bonniot, Université

Joseph Fourier Grenoble I, La Tronche, France

IANPING DONG • Waksman Institute and Department of Chemistry, Howard

Hughes Medical Institute, Rutgers University, Piscataway, NJ

LAUS DOSE • Institut für Biochemie, Johannes Gutenberg-Universität,

Mainz, Germany

RÉGEN DROUIN • Department de Biologie Médicale, Université Laval, et

Unité de Recherche en Génétique Humaine et Moléculaire, Centre de

Recherche, Pavilion Saint-Francois d’Assise,

Québec, Canada

ICHARD H. EBRIGHT • Waksman Institute and Department of Chemistry,

Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ

LISA E. ENGLER • Department of Biological Sciences, University of Pittsburgh,

Pittsburgh, PA

ALEXANDER A. GALL • Seattle Genetics, Bothell, WA

JOHANNES GEISELMANN • Plasticité et Expression des Génomes Microbiens,

Université Joseph Fourier, Grenoble, France

CARLA W. GRAY • Department of Molecular and Cell Biology, University

of Texas at Dallas, Richardson, TX

SYLVAIN GUÉRIN • Laboratory of Molecular Endocrinologie, Centre

Hopitalier Universitaire de Québec, Université Laval, Québec, Canada

JEFFREY J. HAYES • Department of Biochemistry and Biophysics, University of

Rochester Medical Center, Rochester, NY

ERMANN

EUMANN

• Max Planck Institute of Biochemistry, Martinsried, Germany

MARK ISALAN • Laboratory of Molecular Biology, Medical Research Council,

Cambridge, UK

LIAN

ELESAROV

•Biochemisches Institut der Universität Zurich, Zurich, Switzerland

INDA

-J

ACOBSON

• Department of Biological Sciences, University of Pittsburgh

Pittsburgh, PA

BRENDA F. KAHL • Department of Biochemistry and Molecular Biology,

Colorado State University, Fort Collins, CO

YOUNGGYU KIM • Waksman Institute and Department of Chemistry, Howard

Hughes Medical Institute, Rutgers University, Piscataway, NJ

Contributors

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

University of Portsmouth, Portsmouth, UK

NNIE KOLB • Institut Pasteur, Paris, France

MICHAEL R. KURPIEWSKI • Department of Biological Sciences, University

of Pittsburgh, Pittsburgh, PA

IMON LABBÉ • Department of Biological Chemistry, The University of Michigan

Medical School, Ann Arbor, MI

ARC-ANDRÉ LANIEL • Laboratory of Molecular Endocrinologie, Centre

Hopitalier Universitaire de Québec, Université Laval, Québec, Canada

LIVIER LAROCHELLE • Centre de Recherche en Cancérologie, Université

Laval, CHUQ/L´Hotel-Dieu de Québec, Québec, Canada

EBEAU

•

Faculté de Médecine, Illkirch, France

ENOIT LEBLANC • NIDDK, NIH, Bethesda, MD

SIAO-HUI LIU • Department of Biochemistry and Genetics, Medical School,

University of Newcastle upon Tyne, Newcastle upon Tyne, UK

URI L. LYUBCHENKO • Departments of Biology and Microbiology, Arizona

State University, Tempe, AZ

W. M

ANFIELD

• Department of Genetics, University of Leeds, Leeds, UK

AMES

A. M

LELLAN

• Biophysics Laboratories, School of Biological Sciences

University of Portsmouth, Portsmouth, UK

AINER

EFFERT

• Ministerium für Umwelt und Forsten des Landes Rheinland

Pfalz, Mainz, Germany

ILLI METZGER •

Ministerium für Umwelt und Forsten des Landes Rheinland

Pfalz, Mainz, Germany

OM MOSS • Centre de Recherche en Cancérologie et départment de

Biologie Médicale de l’Université Laval, Centre Hopital Universitaire

de Québec, Québec, Canada

IKOLAI NARYSHKIN • Waksman Institute and Department of Chemistry,

Howard Hughes Medical Institute, Rutgers University, Piscataway, NJ

ETER E. NIELSEN • Department of Medical Biochemistry and Genetics,

Laboratory of Biochemistry, The Panum Institute, Copenhagen, Denmark

NTHONY

W. O

LIVER

•

Biophysics Laboratories, School of Biological

Sciences, University of Portsmouth, Portsmouth, UK

TÉPHANE

UELLET

• Department de Biologie Médicale

Université Laval, et

Unité de Recherche en Génétique Humaine et Moléculaire

, Centre de Recherche,

Pavilion Saint-Francois d’Assise, Québec, Canada

THANASIOS

G. P

APAVASSILIOU

• Department of Biochemistry, School of Medi-

cine

, University of Patras, Patras, Greece

AMIAN PARRY • Department of Biochemistry and Genetics, Medical School,

University of Newcastle upon Tyne, Newcastle upon Tyne, UK

MARVIN PAULE • Department of Biochemistry and Molecular Biology, Colo-

rado State University, Fort Collins, CO

IM PERSINGER • Department of Biochemistry and Molecular Biology, School

of Medicine, Southern Illinois University, Carbondale, IL

IMON E. PLYTE • Pharmacia and Upjohn, Milano, Italy

G. P

OIRIER

• Unité Santé et Environment, CHUQ, Pavillon CHUL,

Québec, Canada

GABRIELE RATHGEBER • Merck KGaA, Darmstadt, Germany

CHRISTOPHER M. READ • Biophysics Laboratories, School of Biological Sciences,

University of Portsmouth, Portsmouth, UK

IRGIL

HODIUS

• School of Biochemistry, University of Birmingham,

Birmingham, UK

FRANÇOIS ROBERT • Whitehead Institute for Biomedical Research, Cambridge, MA

HRISTOPH

USSMANN

• Fachbereich Physik, Universität Kaiserlautern, Germany

NIGEL SAVERY • School of Biochemistry, University of Birmingham,

Birmingham, UK

ANS-JOCHEN SCHAFER • Institute für Biochemie, Johannes Gutenberg-Universität,

Mainz, Germany

PETER SCHICKOR • Max Planck Institute of Biochemistry, Martinsried, Germany

EBORAH B. SCHULMAN • Department of Molecular Biology and Microbiology,

School of Medicine, Case Western Reserve University, Cleveland, OH

ATRICK SCHULTZ • Faculté de Médecine, Illkirch, France

ARL

ÉGUIN

•

Centre de Recherche en Cancérologie, Université Laval,

CHUQ/L´Hotel-Dieu de Québec, Québec, Canada

AVID R. SETZER • Department of Molecular Biology and Microbiology,

School of Medicine, Case Western Reserve University, Cleveland, OH

ETER

E. S

HAW

• Department of Biochemistry, School of Biomedical

Sciences,

University of Nottingham, Queen’s Medical Center, Nottingham, UK

UDA

S. S

HLYAKHTENKO

• Departments of Plant Biology and Microbiology,

Arizona State University

, Tempe, AZ

MARTHA N. SIMON • Brookhaven National Laboratory, Biology Department,

Upton, NY

A. F

RANCIS STEWART • European Molecular Biology Laboratory, Heidelberg,

Germany

ALE STEWART • Centre de Recherche en Cancérologie, Université Laval,

CHUQ/L´Hotel-Dieu de Québec, Québec, Canada

ETER

G. S

TOCKLEY

• Department of Genetics, University of Leeds, Leeds, UK

IAN TAYLOR • Laboratory of Molecular Biophysics, University of Oxford,

Oxford, UK

xvi Contributors

Contributors xvii

JEAN-PHILIPPE THERRIEN • Division de Pathologie, Department de Biologie

Médicale, Université Laval, et Unité de Recherche en Génétique Humaine

et Moléculaire, Centre de Recherche, Pavilion Saint-Francois d’Assise,

Québec, Canada

ANDREW A. TRAVERS • Lab Molecular Biology, Medical Research Council,

Cambridge, UK

OSEPH S. WALL • Brookhaven National Laboratory, Biology Department,

Upton, NY

ICHELLE WEBB • Department of Chemistry, University of Sheffield, Sheffield

VGENY

AYCHIKOV

• Max Planck Institute of Biochemistry, Martinried, Germany

CHRISTIAN ZWIEB • Department of Molecular Biology, The University of Texas

Health Center at Tyler, Tyler, TX

Filter-Binding Assays 1

From:

Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.

Filter-Binding Assays

Peter G. Stockley

1. Introduction

Membrane filtration has a long history in the analysis of protein–nucleic

acid complex formation, having first been used to examine RNA–protein inter-

actions (1), before being introduced to DNA–protein interaction studies by

Jones and Berg in 1966 (2). The principle of the technique is straightforward.

Under a wide range of buffer conditions, nucleic acids pass freely through

membrane filters, whereas proteins and their bound ligands are retained. Thus,

if a particular protein binds to a specific DNA sequence, passage through the

filter will result in retention of a fraction of the protein–DNA complex by vir-

tue of the protein component of the complex. The amount of DNA retained can

be determined by using radioactively labeled DNA to form the complex and

then determining the amount of radioactivity retained on the filter by scintilla-

tion counting. The technique can be used to analyze both binding equilibria

and kinetic behavior, and if the DNA samples retained on the filter and in the

filtrate are recovered for further processing, the details of the specific binding

site can be probed by interference techniques.

The technique has a number of advantages over footprinting and gel retarda-

tion assays, although there are also some relative disadvantages, especially

where multiple proteins are binding to the same DNA molecule. However, fil-

ter binding is extremely rapid, reproducible, and, in principle, can be used to

extract accurate equilibrium and rate constants (3–5). We have used the

technique to examine the interaction between the E. coli methionine repressor,

MetJ, and various operator sites cloned into restriction fragments (6,7, see

also Chapter 15). Results from these studies will be used to illustrate the

basic technique.

2Stockley

Before discussing the experimental protocols it is important to understand

some fundamental properties of the filter-binding assay. The molecular basis

of the discrimination between nucleic acids and proteins during filtration is

still not fully understood. Care should therefore be taken to characterize the

assay with the system under study. Nucleic acid–protein complex retention

occurs with differing efficiencies, depending on the lifetime of the complex,

the size of the protein component, the buffer conditions, and the extent of wash-

ing of the filter. Experiments with the lac repressor system have shown that

prior filtration of protein followed by passage of DNA containing operator

sites does not result in significant retention of the nucleic acid, presumably

because filter-bound protein is inactive for further operator binding. The DNA

retained on filters is therefore a direct reflection of the amount of complex

present when filtration began. Furthermore, incubation of the lac repressor with

large amounts of DNA that does not contain an operator site followed by filtra-

tion also does not lead to significant retention. Because the lac repressor (and,

indeed, essentially all DNA-binding proteins) binds nonsequence-specifically

to DNA, forming short-lived complexes, it is clear that these are not readily

retained. The experiments with the lac repressor (3–5) can therefore be used as

a guide when designing experimental protocols. The repressor is a large pro-

tein (being a tetramer of 38-kDa subunits) but the basic features seem to apply

even to short peptides with molecular weights <2 kDa (8).

In any particular system, the percentage of the DNA–protein complex in

solution retained by the filter should ideally be constant throughout the bind-

ing curve, and this is known as the retention efficiency. Experimental values

range from 30 to >95%. An example of the sort of results obtained with the

MetJ repressor is shown in Fig. 1.

2. Materials

2.1. Preparation of Radioactively End-Labeled DNA

1. Plasmid DNA carrying the binding site for a DNA-binding protein on a conve-

nient restriction fragment (usually <200 bp).

2. Restriction enzymes and the appropriate buffers as recommended by the suppliers.

3. Phenol: redistilled phenol equilibrated with 100 mM Tris–HCl, pH 8.0.

4. Chloroform.

5. Solutions for ethanol precipitation of DNA: 4 M NaCl and ethanol (absolute and

70% v/v).

6. Calf intestinal alkaline phosphatase (CIAP).

7. CIAP reaction buffer (10X): 0.5 M Tris–HCl, pH 9.0, 0.01 M MgCl

, 0.001 M ZnCl

8. TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA).

9. 20% w/v Sodium dodecyl sulfate (SDS).

10. 0.25 M EDTA, pH 8.0.

Filter-Binding Assays 3

11. T4 polynucleotide kinase (T4 PNK).

12. T4 PNK reaction buffer, 10X: 0.5 M Tris–HCl, pH 7.6, 0.1 M MgCl

, 0.05 M

dithiothreitol.

13. Radioisotope: γ-[

P]-ATP.

14. 30% w/v acrylamide stock (29:1 acrylamide: N,N'-methylene-bisacrylamide).

15. Polyacrylamide gel elution buffer: 0.3 M sodium acetate, 0.2% w/v sodium

dodecyl sulfate (SDS), 2 mM EDTA.

16. Polymerization catalysts: ammonium persulfate (10% w/v) and N,N,N',N'-

tetramethylethylene diamine (TEMED).

17. X-ray film, autoradiography cassette and film developer.

18. Plastic wrap and scalpel.

2.2. Filter-Binding Assays

1. Nitrocellulose filters: We use HAWP (00024) filters from Millipore (Bedford,

MA), but suitable filters are available from a number of other manufacturers,

such as Schleicher and Schuell (Dassel, Germany). Filters tend to be relatively

expensive. Some manufacturers produce sheets of membrane that can be cut to

size and are thus less expensive.

2. Filter-binding buffer (FB): 100 mM KCl, 0.2 mM EDTA, 10 mM Tris–HCl,

pH 7.6.

3. Binding buffer (BB): This is FB containing 50 µg/mL bovine serum albumin

(BSA, protease and nuclease free; see Note 1).