DNA Repair, Part B (eBook)

Cover Page 1
Table of Contents 6
Contributors to Volume 409 10
Volume in Series 16
Chapter 1: Overproduction and Purification of RFC-Related Clamp Loaders and PCNA-Related Clamps from Saccharomyces cerevisiae 39
Introduction 39
Overproduction of Complexes in Yeast 41
Extract Preparation and Purification 43
Discussion 47
Acknowledgments 48
References 48
Chapter 2: Functional Assays for Replication Protein A (RPA) 49
Introduction 50
Purification of Recombinant RPA 51
DNA-Binding Assays 54
Gel-Binding Assays (Gel Mobility Shift and Helix-Destabilization) 54
Surface Plasmon Resonance 59
Fluorescence Polarization 64
Other DNA-Binding Assays 65
RPA-Protein Interactions (Enzyme-Linked Immunosorbent Assay) 65
Functional Assays for RPA 67
In Vivo Localization 69
In Vivo Localization by Indirect Immunofluorescence 69
Summary and Conclusion 72
References 72
Chapter 3: Human DNA Ligases I, III, and IV-Purification and New Specific Assays for These Enzymes 77
Introduction 77
Overexpression and Purification of Human DNA Ligases 78
DNA Ligation Assays 82
Biotinylated Linear DNA Substrates with Blocked DNA Ends 83
Linear DNA Substrates to Measure Intra- and Intermolecular DNA Joining 84
Ligation Assays Using Fluorescent DNA Substrates 85
Concluding Remarks 87
Acknowledgments 88
References 88
Chapter 4: Enzymatic Mechanism of the WRN Helicase/Nuclease 90
Expression and Purification of Recombinant WRN Protein 91
Characterization of WRN Helicase Activity 92
Helicase Assays 95
Preparation of Radiolabeled Duplex DNA Substrate 96
Preparation of Four-Stranded Oligonucleotide-Based Holliday Junction 97
Preparation of Three-Stranded Oligonucleotide-Based D-Loop 98
Radiometric Helicase Assay 100
Real-Time Fluorometric Helicase Assay 102
ATPase Assay 105
Gel Mobility Shift Assay to Measure DNA Binding 107
Exonuclease Assay 109
Effects of Post-Translational Modifications on WRN Catalytic Activities 116
WRN Protein Interactions that Modulate WRN Catalytic Activity 118
Acknowledgments 120
References 120
Chapter 5: Analysis of the DNA Unwinding Activity of RecQ Family Helicases 124
Introduction 124
Preparation of Substrates 125
Analysis of Helicase Activity 132
Safety Issues 136
References 137
Chapter 6: Characterization of Checkpoint Responses to DNA Damage in Saccharomyces cerevisiae: Basic Protocols 139
General Introduction 139
Budding Analysis 140
Microcolony Assay 143
Visualization of the Mitotic Spindle 145
Nuclear Staining with DAPI 148
Flow Cytometry 149
Analysis of Rad53 Phosphorylation 152
Acknowledgments 154
References 154
Chapter 7: Recruitment of ATR-ATRIP, Rad17, and 9-1-1 Complexes to DNA Damage 156
Introduction 156
Chromatin Association of ATR, Rad17, Rad9, and RPA 158
Purification of ATR, ATRIP, and Reconstitution of the ATR-ATRIP Complex 160
Recruitment of ATRIP and ATR-ATRIP to RPA-Coated Single-Stranded DNA 161
Purification of Rad17 and 9-1-1 Complexes 163
Recruitment of 9-1-1 Complexes to Primed ssDNA by RPA and Rad17 Complexes 164
Conclusion 166
Acknowledgments 167
References 167
Chapter 8: Multiple Approaches to Study S. cerevisiae Rad9, a Prototypical Checkpoint Protein 169
Introduction 169
Cell Cycle Checkpoint Analysis 171
Summary 185
Acknowledgments 186
References 186
Chapter 9: Methods for Studying Adaptation to the DNA Damage Checkpoint in Yeast 188
Introduction 188
Designing Adaptation Assays 189
Ensuring that DSBs Are Irreparable 190
IR-Induced DSBs 191
HO-Induced DSBs 192
Using Disomic Strains 194
Adaptation Assays Based on Chromosome Loss 199
Adaptation to cdc13-Induced Damage 201
Conclusions 202
References 203
Chapter 10: DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae 204
Introduction 204
Methods 205
Purification of Rad55-Rad57 Heterodimer from S. cerevisiae 213
In Vitro Kinase Assays 215
Conclusions and Perspectives 219
Acknowledgments 219
References 220
Chapter 11: Methods for Studying Mutagenesis and Checkpoints in Schizosaccharomyces pombe 221
Introduction 221
Protocol 222
Protocol 227
References 231
Chapter 12: Methods for Determining Spontaneous Mutation Rates 233
Introduction 233
Terminology 234
The Lea-Coulson Model 235
Mutant Accumulation 235
Fluctuation Analysis 237
Acknowledgments 249
References 249
Chapter 13: Genomic Approaches for Identifying DNA Damage Response Pathways in S. cerevisiae 251
Introduction 251
Systematic Genome-Wide Screens to Identify Genes Required for Resistance to DNA Damaging Agents 252
Synthetic Genetic Array Analysis to Identify DNA Damage Response Pathways 260
Acknowledgments 271
References 271
Chapter 14: Techniques for gamma-H2AX Detection 274
Introduction 274
Sources of Antibodies to gamma-H2AX 274
Immunocytochemical Detection of gamma-H2AX Foci in Mammalian Material 275
Combined Immunocytochemistry and FISH on Metaphase Spreads 279
Immunocytochemical Detection of gamma-H2AX Foci in Budding Yeast 282
Immunoblot Detection of Yeast gamma-H2A 285
Chromatin Immunoprecipitation Using Yeast Anti-gamma-H2AX 286
References 288
Further Reading 288
Chapter 15: Methods for Studying the Cellular Response to DNA Damage: Influence of the Mre11 Complex on Chromosome Metabolism 289
Introduction 289
Derivation of Primary and Transformed Cells 290
IR-Induced Cell Cycle Checkpoints 294
Metaphase Spreads 307
Immunofluorescence 314
Acknowledgments 321
References 321
Chapter 16: Detecting Repair Intermediates In Vivo: Effects of DNA Damage Response Genes on Single-Stranded DNA Accumulation at Uncapped Telomeres in Budding Yeast 323
Introduction 323
Experimental Outline 324
Methods 325
Acknowledgments 338
References 338
Chapter 17: Analysis of Non-B DNA Structure at Chromosomal Sites in the Mammalian Genome 339
Introduction 339
Specific Methods 340
Potassium Permanganate and Osmium Tetroxide Chemical Probing 347
Conclusion 351
Acknowledgments 352
References 352
Chapter 18: Detection and Structural Analysis of R-Loops 354
Introduction 355
Procedures 356
References 366
Chapter 19: The Delitto Perfetto Approach to In Vivo Site-Directed Mutagenesis and Chromosome Rearrangements with Synthetic Oligonucleotides in Yeast 367
Overview 367
Creation and Utility of Delitto Perfetto Systems 371
Conclusions 382
References 382
Chapter 20: Assays for Transcriptional Mutagenesis in Active Genes 383
Introduction 384
ssDNA Production 386
Synthesis of the Transcribed Strand 387
Purification of Closed Circular DNA Molecules 389
Quantification of Transcriptional Mutagenesis 390
Determining the Transcriptional Mutagenesis Spectrum 392
Conclusions 394
Acknowledgments 395
References 395
Chapter 21: Methods for Studying Chromatin Assembly Coupled to DNA Repair 396
Introduction 396
Analysis of Chromatin Assembly Coupled to DNA Repair In Vitro 398
Analysis of Chromatin Assembly Coupled to DNA Repair In Vivo 406
Conclusion and Perspectives 409
Acknowledgments 410
References 410
Chapter 22: Structure-Function Analysis of SWI2/SNF2 Enzymes 413
Introduction 413
Biochemical Characterization of SWI2/SNF2 ATPases 414
DNA Translocation Monitoring-Triplex Displacement Assay 416
Detection of Superhelical Torsion-Cruciform Extrusion Assay 418
Crystallization of Protein:DNA Complexes 420
Conclusions 425
Acknowledgments 425
References 425
Chapter 23: Genomic Approach for the Understanding of Dynamic Aspect of Chromosome Behavior 427
Introduction 427
Outline of ChIP-Chip Technique 428
ChIP-Chip Protocol for the Analysis of Protein Binding Profile in S-Phase 429
Solutions for ChIP-chip Analysis 430
Location Analyses of DNA Replication Proteins and Replicated Regions 445
Acknowledgments 448
Reference 448
Chapter 24: Measurement of Chromosomal DNA Single-Strand Breaks and Replication Fork Progression Rates 448
Measurement of Chromosomal Single-Strand Breaks by Alkaline Single-Cell Agarose-Gel Electrophoresis (Alkaline Comet Assay) 449
Identification of Novel SSBR Polypeptides by Yeast 2-Hybrid Library Screens 453
Recovery of XRCC1 Protein Complexes from Cell Extract by Immobilized Metal-Chelate Chromatography (IMAC) 455
Quantification of Chromosomal Replication Fork Rates on Damaged DNA by DNA Fiber Labeling 458
References 463
Chapter 25: Monitoring DNA Replication Following UV-Induced Damage in Escherichia coli 463
Introduction 463
Description of the Methods and Technical Comments 465
Concluding Remarks 477
Acknowledgments 477
References 478
Chapter 26: Methods to Study Replication Fork Collapse in Budding Yeast 480
Introduction 480
Methods 481
Cell Background 493
Acknowledgments 499
References 499
Chapter 27: Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae 500
Introduction 501
Selection of Yeast Cells with Gross-Chromosomal Rearrangements 503
Determining the Rate of Accumulating Gross-Chromosomal Rearrangements 504
Mapping and Sequencing of Chromosome V Breakpoints 506
Chromosome V Rearrangement Types 510
Conclusion 512
References 514
Chapter 28: Formation and Processing of Stalled Replication Forks-Utility of Two-Dimensional Agarose Gels 515
Introduction 515
Protocol 517
Interpretation 519
Closing Comments 527
Acknowledgments 529
References 529
Chapter 29: Poly(ADP-ribose) Polymerase-1 Activation During DNA Damage and Repair 531
Affinity Purification of Recombinant DNA-Damage Dependent Poly(ADP-Ribose) Polymerases (PARPs) 532
PARP-1 Activation and DNA Damage 535
PARP-1 and DNA Repair 538
Acknowledgments 547
References 547
Chapter 30: Tyrosyl-DNA Phosphodiesterase (Tdp1) (3'-Phosphotyrosyl DNA Phosphodiesterase) 549
Introduction 549
Tdp1 550
DNA Substrates 551
Kinetic Analysis 556
DNA Binding 559
References 561
Chapter 31: Assaying Double-Strand Break Repair Pathway Choice in Mammalian Cells Using a Targeted Endonuclease or the RAG Recombinase 562
Introduction 563
Materials 568
Methods 570
Notes 577
Acknowledgments 577
References 577
Author Index 579
Subject Index 601