DNA Repair

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Introduction......Page 12
Expression Plasmids and Strains......Page 14
Procedure......Page 15
Breakage of Cells with Dry Ice......Page 16
Glutathione Sepharose Chromatography......Page 17
Purification of Ctf18-RFC and Elg1-RFC......Page 19
Discussion......Page 20
References......Page 21
Functional Assays for Replication Protein A (RPA)......Page 23
Introduction......Page 24
Buffers......Page 25
Extract Preparation......Page 26
Mono‐Q Chromatography......Page 27
Gel-Binding Assays (Gel Mobility Shift and Helix-Destabilization)......Page 28
GMSA Assay......Page 30
Helix Destabilization Assay......Page 32
Surface Plasmon Resonance......Page 33
Surface Plasmon Resonance Procedure......Page 34
Fluorescence Polarization......Page 38
RPA-Protein Interactions (Enzyme-Linked Immunosorbent Assay)......Page 39
Experimental Procedure......Page 40
DNA Replication......Page 41
DNA Repair Assays......Page 42
Buffers......Page 43
Methods......Page 44
Methods......Page 45
References......Page 46
Introduction......Page 51
Overexpression and Purification of Human DNA Ligases......Page 52
DNA Ligation Assays......Page 56
Biotinylated Linear DNA Substrates with Blocked DNA Ends......Page 57
Linear DNA Substrates to Measure Intra and Intermolecular DNA Joining......Page 58
Ligation Assays Using Fluorescent DNA Substrates......Page 59
Concluding Remarks......Page 61
References......Page 62
Enzymatic Mechanism of the WRN Helicase/Nuclease......Page 65
WRN Bacmid Construction and Baculovirus Infection......Page 66
Directionality and Loading Properties of WRN Helicase......Page 67
Length Dependence for Unwinding by WRN Helicase......Page 69
Helicase Assays......Page 70
Preparation of Radiolabeled Duplex DNA Substrate......Page 71
Preparation of Four-Stranded Oligonucleotide-Based Holliday Junction......Page 72
Preparation of Three-Stranded Oligonucleotide-Based D-Loop......Page 73
Radiometric Helicase Assay......Page 75
Real-Time Fluorometric Helicase Assay......Page 77
ATPase Assay......Page 80
Gel Mobility Shift Assay to Measure DNA Binding......Page 82
Substrate Specificity of WRN Exonuclease......Page 84
Radiometric Exonuclease Assay......Page 86
Independent Analysis of WRN Exonuclease and Helicase Activities......Page 89
Combined Analysis of WRN Helicase and Exonuclease Activities......Page 90
Effects of Post-Translational Modifications on WRN Catalytic Activities......Page 91
WRN Protein Interactions that Modulate WRN Catalytic Activity......Page 93
References......Page 95
Introduction......Page 99
Substrates Prepared from Annealed Oligonucleotides......Page 100
Procedure......Page 102
Materials......Page 103
Generation of G4 Quadruplex Substrates......Page 104
Procedure......Page 105
The Assay System......Page 107
Procedure 1......Page 109
Procedure 2......Page 110
Safety Issues......Page 111
References......Page 112
Further Reading......Page 113
General Introduction......Page 114
Protocol......Page 115
Protocol......Page 118
Introduction......Page 120
Protocol......Page 122
Introduction......Page 123
Introduction......Page 124
Materials......Page 125
Introduction......Page 127
Yeast Protein Preparation......Page 128
References......Page 129
Introduction......Page 131
Chromatin Association of ATR, Rad17, Rad9, and RPA......Page 133
Purification of ATR, ATRIP, and Reconstitution of the ATR-ATRIP Complex......Page 135
Recruitment of ATRIP and ATR-ATRIP to RPA-Coated Single-Stranded DNA......Page 136
Purification of Rad17 and 9-1-1 Complexes......Page 138
Recruitment of 9-1-1 Complexes to Primed ssDNA by RPA and Rad17 Complexes......Page 139
Conclusion......Page 141
References......Page 142
Introduction......Page 145
Cell Cycle Checkpoint Analysis......Page 147
Analysis of Rad9 Phospho-Forms by Western Blotting......Page 154
Rad53 Release and Catalysis Assays......Page 155
Fractionation of Soluble and Chromatin-Associated Proteins......Page 157
Live Cell Imaging......Page 159
Summary......Page 161
References......Page 162
Introduction......Page 165
Designing Adaptation Assays......Page 166
Ensuring that DSBs Are Irreparable......Page 167
IR-Induced DSBs......Page 168
HO-Induced DSBs......Page 169
Using Disomic Strains......Page 171
Adaptation Assays Based on Chromosome Loss......Page 176
Adaptation to cdc13-Induced Damage......Page 178
Conclusions......Page 179
References......Page 180
Introduction......Page 181
Analysis of Rad55 Phosphorylation Status in Response to DNA Damage......Page 182
Reagents......Page 183
Procedure......Page 184
Comments to Protocol 1......Page 187
Vector System to Express Heterodimeric Proteins in S. cerevisiae......Page 188
Protocol 2: Purification of Rad55-Rad57 Heterodimers......Page 190
Procedure......Page 191
Protocol 3: Pull-Down of GST-Tagged Rad53 Kinase......Page 192
Procedure......Page 193
Comments to Protocol 3......Page 194
Procedure......Page 195
Acknowledgments......Page 196
References......Page 197
Introduction......Page 198
Mutator Assay......Page 199
Intrachromosomal Recombination Assay......Page 201
Checkpoint Responses to Replication Stalling and DNA Damage......Page 202
Assay of Cds1 Kinase Activity......Page 204
Assay of Chk1 Activation......Page 205
Chromatin Fractionation Assay......Page 206
Checkpoint Regulation of Mutagenesis......Page 207
References......Page 208
Introduction......Page 210
Terminology......Page 211
Mutant Accumulation......Page 212
Experimental Design......Page 214
Analyzing the Results of Fluctuation Assays......Page 217
Calculating the Mutation Rate......Page 222
Confidence Limits for p0......Page 223
Confidence Limits for m Obtained from the MSS Maximum Likelihood Method......Page 224
Phenotype Lag......Page 225
References......Page 226
Introduction......Page 229
Array-Based Screening......Page 230
Pinning Procedure......Page 232
Robotic Pinning Systems......Page 233
Barcode-Based Screening......Page 234
Protocol for Barcode-Based Chemical-Genetic Screening......Page 236
Synthetic Genetic Array Analysis to Identify DNA Damage Response Pathways......Page 238
Clustering Analysis......Page 239
How to Use Cluster 3.0 and Java Treeview......Page 241
Genome-Wide Screens for Suppressors of Genomic Instability......Page 244
Using SGA to Cross Reporters into Deletion Mutant Arrays......Page 246
Screening Using a Patch Assay......Page 247
Media Recipes......Page 248
References......Page 249
Sources of Antibodies to gamma-H2AX......Page 252
Immunocytochemical Detection of gamma-H2AX Foci in Mammalian Material......Page 253
Preparation of Tissue Touchprints......Page 255
Immunocytochemistry, Single Label......Page 256
Combined Immunocytochemistry and FISH on Metaphase Spreads......Page 257
Immunocytochemistry gamma-H2AX on Metaphase Spreads (FISH Compatible)......Page 258
Telomere FISH on Metaphase Spreads......Page 259
Immunocytochemical Detection of gamma-H2AX Foci in Budding Yeast......Page 260
Formation of DNA DSBs in Budding Yeast......Page 261
Visualization of gamma-foci in Budding Yeast......Page 262
Protein Extraction......Page 263
Chromatin Immunoprecipitation Using Yeast Anti-gamma-H2AX......Page 264
Further Reading......Page 266
Introduction......Page 267
Method......Page 268
Materials......Page 269
Materials......Page 270
Method......Page 271
IR-Induced Cell Cycle Checkpoints......Page 272
Materials......Page 273
Method......Page 274
Data Analysis......Page 275
Materials......Page 278
Method......Page 279
Data Analysis......Page 280
Introduction......Page 282
controls......Page 283
Method......Page 284
Material......Page 285
Method for Preparing Cells......Page 287
Method for Fixation, Slide Preparation, and Staining......Page 288
Statistical Concepts......Page 289
Data Analysis......Page 290
Immunofluorescence......Page 292
Controls......Page 293
Materials......Page 294
Method......Page 295
Materials......Page 297
Data Analysis......Page 298
References......Page 299
Introduction......Page 301
Experimental Outline......Page 302
Plaque Preparation and Testing......Page 303
Sample Collection During Incubation at 36deg......Page 304
Culture Preparation and Inoculation......Page 305
Determination of Cell Viability......Page 306
Buffers/Solutions Needed......Page 307
Cell Breakage......Page 308
DNA Elution......Page 309
Adding PCR Master Mix to DNA......Page 310
Analysis of Real-Time PCR Data and Concentration Adjustment......Page 312
Measuring ssDNA by QAOS......Page 314
Mixing Native and Boiled DNA......Page 315
References......Page 316
Introduction......Page 317
Chemistry......Page 318
Procedure......Page 321
Precautions......Page 323
Full Molecule Bisulfite Modification Assay......Page 324
Potassium Permanganate and Osmium Tetroxide Chemical Probing......Page 325
Procedure......Page 326
Conclusion......Page 329
References......Page 330
Detection and Structural Analysis of R-Loops......Page 333
Introduction......Page 334
Precautions......Page 335
Discussion......Page 337
Structural Analysis of the R-Loop with Native Sodium Bisulfite Probing and DNA Sequencing......Page 338
Discussion......Page 340
Procedure......Page 341
Discussion......Page 344
References......Page 345
Overview......Page 347
CORE Cassettes......Page 351
Transformation Protocol......Page 353
Colony PCR Reaction Conditions to Identify Desired Isolates......Page 354
Protocol for Transformation with Oligonucleotide......Page 355
Protocol for Targeting Oligonucleotides to a Region Containing a DSB......Page 357
Break-Mediated Delitto Perfetto with Oligonucleotides in Diploid Cells......Page 358
Generation of Chromosomal Rearrangements......Page 359
References......Page 362
Assays for Transcriptional Mutagenesis in Active Genes......Page 364
Introduction......Page 365
ssDNA Production......Page 367
Second Strand Synthesis......Page 368
Purification of Closed Circular DNA Molecules......Page 370
Preparation of Competent E. coli Cells and Transformation Conditions......Page 371
Luciferase Assay......Page 372
RNA Extraction and RT-PCR......Page 373
Sub-Cloning of RT-PCR Product......Page 374
Conclusions......Page 375
References......Page 376
Introduction......Page 377
S100 Extract......Page 379
Nuclear Extracts......Page 380
Plasmid Labeling and Supercoiling Assay: Nucleosome Assembly Associated with DNA Synthesis (Fig. 1)......Page 381
Histone Deposition on Immobilized DNA (Fig. 2)......Page 383
Controls......Page 386
Global Recruitment of Repair and Chromatin Assembly Factors to Damaged Chromatin (Fig. 3)......Page 387
Monitoring Chromatin Assembly Locally at Damage Sites (Fig. 4)......Page 388
Conclusion and Perspectives......Page 390
References......Page 391
Introduction......Page 394
ATPase Activity......Page 395
Example Protocols for DNA Dependent ATPase Assay......Page 396
DNA Translocation Monitoring-Triplex Displacement Assay......Page 397
Preparation of the Triplex Substrate......Page 398
Detection of Superhelical Torsion-Cruciform Extrusion Assay......Page 399
Protocol for Monitoring the Generation of Altered Superhelical Torsion by Cruciform Extrusion......Page 400
Protein......Page 401
DNA Design......Page 402
Preparation of the Protein:DNA Complex......Page 404
Trapping of Conformational States......Page 405
References......Page 406
Introduction......Page 408
Outline of ChIP-Chip Technique......Page 409
ChIP-Chip Protocol for the Analysis of Protein Binding Profile in S-Phase......Page 410
Extract Preparation......Page 411
Washes......Page 412
DNA Amplification......Page 413
Hybridization......Page 415
Discrimination Analysis......Page 416
Alternative Protocol for DNA Amplification (IVT Amplification Method)......Page 418
Materials......Page 419
Location Analyses of DNA Replication Proteins and Replicated Regions......Page 426
References......Page 429
Measurement of Chromosomal DNA Single-Strand Breaks and Replication Fork Progression Rates......Page 430
General Considerations......Page 431
Materials......Page 432
Protocol......Page 433
Data Acquisition and Analysis......Page 434
General Considerations......Page 435
Protocol......Page 436
Introduction and Overview......Page 437
General Considerations......Page 438
Protocol......Page 439
General Considerations......Page 440
Solutions......Page 441
Labeling and Spreading......Page 442
Immunostaining......Page 443
Image Analysis......Page 444
References......Page 445
Introduction......Page 446
General Considerations for Cell Culture and UV Irradiation......Page 448
UV Treatment and Recovery Assay......Page 449
Sample Preparation and Analysis of Recovery......Page 451
DNA Degradation at Arrested Replication Forks......Page 452
UV Treatment and Degradation Assay......Page 453
Sample Preparation and Analysis of Degradation......Page 454
Plasmid Replication Intermediates Observed by 2D N/N Gel Analysis......Page 455
UV Irradiation and DNA Isolation......Page 456
Two-Dimensional Agarose Gel Electrophoresis......Page 458
Acknowledgments......Page 460
References......Page 461
Introduction......Page 463
Analysis of Stalled and Collapsed Replication Forks Using 2D Gel Electrophoresis......Page 464
Materials and Solutions......Page 466
Supernatant......Page 467
Materials and Solutions......Page 468
Procedure......Page 469
First and Second Dimension Gel Electrophoresis and Southern Hybridization......Page 470
Branch Migration of Joint Molecules......Page 471
Preparation of In Vivo-Crosslinked DNA Samples for Electron Microscopy Analysis......Page 473
Purification of Replication Intermediates by BND Cellulose Column Chromatography......Page 474
Purification of Replication Intermediates by Size-Exclusion Column......Page 475
Cell Background......Page 476
DNA Labeling......Page 477
Materials and Solutions......Page 478
Materials and Solutions......Page 479
Procedure......Page 480
Procedure......Page 481
References......Page 482
Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae......Page 484
Introduction......Page 485
Selection of Yeast Cells with Gross-Chromosomal Rearrangements......Page 487
Determining the Rate of Accumulating Gross-Chromosomal Rearrangements......Page 488
Breakpoint Mapping......Page 490
Breakpoint Junction Amplification and Sequencing......Page 492
Chromosome V Rearrangement Types......Page 494
Conclusion......Page 496
References......Page 498
Introduction......Page 499
Cell Manipulations......Page 501
Southern Blot, Using Sponge Downward Transfer......Page 502
Interpretation......Page 503
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 505
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 507
Examples of 2D Gels with Blocked Replication Forks......Page 508
In Vivo Processing of Blocked Replication Forks......Page 510
Closing Comments......Page 511
References......Page 513
Poly(ADP-ribose) Polymerase-1 Activation During DNA Damage and Repair......Page 516
Methods......Page 517
PARP-1 Purification Protocol from Insect Cells......Page 518
ECH Sepharose 4B-3AB Affinity Purification of PARP-1......Page 519
Detection of PARP-1 Activity in Activity Blots......Page 520
Materials......Page 521
Protocol (for One Plate)......Page 522
Base Excision Repair Assay......Page 523
Materials......Page 524
Procedure. Phosphorylation of oligonucleotides......Page 525
Procedure......Page 526
Repair Assay......Page 527
Purification of the DNA Product......Page 529
Solutions......Page 530
Poly(ADP-Ribose) Synthesis and XRCC1 Recruitment at Laser Induced DNA Strand Breaks......Page 531
References......Page 532
Introduction......Page 534
Tdp1......Page 535
3'-Phosphotyrosine DNA......Page 536
3'-(4-Nitro)Phenyl Phosphate DNA......Page 539
3'-(4-Methylumbelliferone) Phosphate DNA......Page 540
Kinetic Analysis......Page 541
DNA Binding......Page 544
References......Page 546
Assaying Double-Strand Break Repair Pathway Choice in Mammalian Cells Using a Targeted Endonuclease or the RAG Reco.........Page 548
Measuring the Nature and Frequency of Repair of DNA Double-Strand Breaks Generated by the I-SceI Endonuclease......Page 549
Measuring the Nature and Frequency of Repair of DSBs Generated by the RAG Recombinase......Page 551
Site Loss PCR......Page 554
PCR-Southern Assay......Page 555
Measuring HR of a Chromosomal I-SceI DSB......Page 556
Measuring the Frequency of Imprecise NHEJ of an I-SceI-Generated DSB......Page 557
Assaying the Frequency of SSA of an I-SceI-Generated DSB Using the DR-GFP Reporter......Page 558
Analyzing Individual Repair Products......Page 559
Measuring SSA Using a Fluorescence Assay......Page 560
Determining the Frequency of V(D)J Recombination in a Chromosomal Substrate......Page 561
Assaying SSA of a RAG-Induced Chromosomal Break......Page 562
References......Page 563