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Assembles a collection of some 100 techniques for identifying key protein-DNA interactions and defining their components, their manner of interaction, and their manner of function, both in the cell and in the test tube. Techniques span a wide range, from factor identification to atomic detail, and include multiple DNA footprinting analyses, gel shift (EMSA) optimization, SELEX, surface plasmon resonance, site-specific DNA-protein crosslinking, and UV laser crosslinking. For biochemists and molecular, cellular, and developmental biologists. Moss is affiliated with the Center for Cancer Research at the University of Laval, France.

Book Info

Univ. Laval, Quebec, Canada. Offers an array of more than 100 current and reproducible techniques biochemists and molecular, cellular, and developmental biologists can use to illuminate DNA-protein interactions. Includes protocols for in vivo footprinting. Hardcover, softcover not available. -This text refers to an out of print or unavailable edition of this title.

Preface v
Contributors xiii
1 Filter-Binding Assays 1
2 Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions 13
3 DNase I Footprinting 31
4 Footprinting with Exonuclease III 39
5 Hydroxyl Radical Footprinting 49
6 The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA 63
7 Footprinting DNA-Protein Interactions in Native Polyacrylamide Gels by Chemical Nucleolytic Activity of 1, 10-Phenanthroline-Copper 77
8 Uranyl Photofootprinting 111
9 Osmium Tetroxide Modification and the Study of DNA-Protein Interactions 121
10 Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots 135
11 Diffusible Singlet Oxygen as a Probe of DNA Deformation 151
12 Ultraviolet-Laser Footprinting 161
13 In Vivo DNA Analysis 175
14 Identification of Protein-DNA Contacts with Dimethyl Sulfate: Methylation Protection and Methylation Interference 221
15 Ethylation Interference 229
16 Hydroxyl Radical Interference 245
17 Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting 255
18 A Competition Assay for DNA Binding Using the Fluorescent Probe ANS 265
19 Site-Directed Cleavage of DNA by Linker Histone-Fe(ll) EDTA Conjugates 275
20 Nitration of Tyrosine Residues in Protein-Nucleic Acid Complexes 291
21 Chemical Modification of Lysine by Reductive Methylation: A Probe of Residues Involved in DNA Binding 301
22 Limited Proteolysis of Protein-Nucleic Acid Complexes 315
23 Ultraviolet Crosslinking of DNA-Protein Complexes via 8-Azidoadenine 323
24 Site-Specific Protein-DNA Photocrosslinking: Analysis of Bacterial Transcription Initiation Complexes 337
25 Site-Directed DNA Photoaffinity Labeling of RNA Polymerase III Transcription Complexes 363
26 Use of Site-Specific Protein-DNA Photocrosslinking to Analyze the Molecular Organization of the RNA Polymerase II Initiation Complex 383
27 UV Laser-Induced Protein-DNA Crosslinking 395
28 Plasmid Vectors for the Analysis of Protein-Induced DNA Bending 403
29 Engineering Nucleic Acid-Binding Proteins by Phage Display 417
30 Genetic Analysis of DNA-Protein Interactions Using a Reporter Gene Assay in Yeast 431
31 Assays for Transcription Factor Activity 451
32 Assay of Restriction Endonucleases Using Oligonucleotides 465
33 Analysis of DNA-Protein Interactions by Intrinsic Fluorescence 491
34 Circular Dichroism for the Analysis of Protein-DNA Interactions 503
35 Calorimetry of Protein-DNA Complexes and Their Components 511
36 Surface Plasmon Resonance Applied to DNA-Protein Complexes 535
37 Reconstitution of Protein-DNA Complexes for Crystallization 547
38 Two-Dimensional Crystallization of Soluble Protein Complexes 557
39 Atomic Force Microscopy of DNA and Protein-DNA Complexes Using Functionalized Mica Substrates 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 579
41 Scanning Transmission Electron Microscopy of DNA-Protein Complexes 589
42 Determination of Nucleic Acid Recognition Sequences by SELEX 603
43 High DNA-Protein Crosslinking Yield with Two-Wavelength Femtosecond Laser Irradiation 611
Appendices
Appendix I EMSA/Gel Shift Conditions 617
Appendix II DNA-Modification/Cleavage Reagents 619
Index 621