DNA repair

Introduction......Page 35
Expression Plasmids and Strains......Page 37
Procedure......Page 38
Breakage of Cells with Dry Ice......Page 39
Glutathione Sepharose Chromatography......Page 40
Purification of Ctf18-RFC and Elg1-RFC......Page 42
Discussion......Page 43
References......Page 44
Functional Assays for Replication Protein A (RPA)......Page 46
Introduction......Page 47
Buffers......Page 48
Extract Preparation......Page 49
Mono‐Q Chromatography\0......Page 50
Gel-Binding Assays (Gel Mobility Shift and Helix-Destabilization)......Page 51
GMSA Assay......Page 53
Helix Destabilization Assay......Page 55
Surface Plasmon Resonance......Page 56
Surface Plasmon Resonance Procedure......Page 57
Fluorescence Polarization......Page 61
RPA-Protein Interactions (Enzyme-Linked Immunosorbent Assay)......Page 62
Experimental Procedure......Page 63
DNA Replication......Page 64
DNA Repair Assays......Page 65
Buffers......Page 66
Methods......Page 67
Methods......Page 68
References......Page 69
Introduction......Page 74
Overexpression and Purification of Human DNA Ligases......Page 75
DNA Ligation Assays......Page 79
Biotinylated Linear DNA Substrates with Blocked DNA Ends......Page 80
Linear DNA Substrates to Measure Intra and Intermolecular DNA Joining......Page 81
Ligation Assays Using Fluorescent DNA Substrates......Page 82
Concluding Remarks......Page 84
References......Page 85
Enzymatic Mechanism of the WRN Helicase/Nuclease......Page 88
WRN Bacmid Construction and Baculovirus Infection......Page 89
Directionality and Loading Properties of WRN Helicase......Page 90
Length Dependence for Unwinding by WRN Helicase......Page 92
Helicase Assays......Page 93
Preparation of Radiolabeled Duplex DNA Substrate......Page 94
Preparation of Four-Stranded Oligonucleotide-Based Holliday Junction......Page 95
Preparation of Three-Stranded Oligonucleotide-Based D-Loop......Page 96
Radiometric Helicase Assay......Page 98
Real-Time Fluorometric Helicase Assay......Page 100
ATPase Assay......Page 103
Gel Mobility Shift Assay to Measure DNA Binding......Page 105
Substrate Specificity of WRN Exonuclease......Page 107
Radiometric Exonuclease Assay......Page 109
Independent Analysis of WRN Exonuclease and Helicase Activities......Page 112
Combined Analysis of WRN Helicase and Exonuclease Activities......Page 113
Effects of Post-Translational Modifications on WRN Catalytic Activities......Page 114
WRN Protein Interactions that Modulate WRN Catalytic Activity......Page 116
References......Page 118
Introduction......Page 122
Substrates Prepared from Annealed Oligonucleotides......Page 123
Procedure......Page 125
Materials......Page 126
Generation of G4 Quadruplex Substrates......Page 127
Procedure......Page 128
The Assay System......Page 130
Procedure 1......Page 132
Procedure 2......Page 133
Safety Issues......Page 134
References......Page 135
Further Reading......Page 136
General Introduction......Page 137
Protocol......Page 138
Protocol......Page 141
Introduction......Page 143
Protocol......Page 145
Introduction......Page 146
Introduction......Page 147
Materials......Page 148
Introduction......Page 150
Yeast Protein Preparation......Page 151
References......Page 152
Introduction......Page 154
Chromatin Association of ATR, Rad17, Rad9, and RPA......Page 156
Purification of ATR, ATRIP, and Reconstitution of the ATR-ATRIP Complex......Page 158
Recruitment of ATRIP and ATR-ATRIP to RPA-Coated Single-Stranded DNA......Page 159
Purification of Rad17 and 9-1-1 Complexes......Page 161
Recruitment of 9-1-1 Complexes to Primed ssDNA by RPA and Rad17 Complexes......Page 162
Conclusion......Page 164
References......Page 165
Introduction......Page 168
Cell Cycle Checkpoint Analysis......Page 170
Analysis of Rad9 Phospho-Forms by Western Blotting......Page 177
Rad53 Release and Catalysis Assays......Page 178
Fractionation of Soluble and Chromatin-Associated Proteins......Page 180
Live Cell Imaging......Page 182
Summary......Page 184
References......Page 185
Introduction......Page 188
Designing Adaptation Assays......Page 189
Ensuring that DSBs Are Irreparable......Page 190
IR-Induced DSBs......Page 191
HO-Induced DSBs......Page 192
Using Disomic Strains......Page 194
Adaptation Assays Based on Chromosome Loss......Page 199
Adaptation to cdc13-Induced Damage......Page 201
Conclusions......Page 202
References......Page 203
Introduction......Page 204
Cell Manipulations......Page 206
Southern Blot, Using Sponge Downward Transfer......Page 207
Interpretation......Page 208
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 210
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 212
Examples of 2D Gels with Blocked Replication Forks......Page 213
In Vivo Processing of Blocked Replication Forks......Page 215
Closing Comments......Page 216
References......Page 218
Poly(ADP-ribose) Polymerase-1 Activation During DNA Damage and Repair......Page 221
Methods......Page 222
PARP-1 Purification Protocol from Insect Cells......Page 223
ECH Sepharose 4B-3AB Affinity Purification of PARP-1......Page 224
Detection of PARP-1 Activity in Activity Blots......Page 225
Materials......Page 226
Protocol (for One Plate)......Page 227
Base Excision Repair Assay......Page 228
Materials......Page 229
Procedure. Phosphorylation of oligonucleotides......Page 230
Procedure......Page 231
Repair Assay......Page 232
Purification of the DNA Product......Page 234
Solutions......Page 235
Poly(ADP-Ribose) Synthesis and XRCC1 Recruitment at Laser Induced DNA Strand Breaks......Page 236
References......Page 237
Introduction......Page 239
Tdp1......Page 240
3\'-Phosphotyrosine DNA......Page 241
3\'-(4-Nitro)Phenyl Phosphate DNA......Page 244
3\'-(4-Methylumbelliferone) Phosphate DNA......Page 245
Kinetic Analysis......Page 246
DNA Binding......Page 249
References......Page 251
Assaying Double-Strand Break Repair Pathway Choice in Mammalian Cells Using a Targeted Endonuclease or the RAG Reco.........Page 253
Measuring the Nature and Frequency of Repair of DNA Double-Strand Breaks Generated by the I-SceI Endonuclease......Page 254
Measuring the Nature and Frequency of Repair of DSBs Generated by the RAG Recombinase......Page 256
Site Loss PCR......Page 259
PCR-Southern Assay......Page 260
Measuring HR of a Chromosomal I-SceI DSB......Page 261
Measuring the Frequency of Imprecise NHEJ of an I-SceI-Generated DSB......Page 262
Assaying the Frequency of SSA of an I-SceI-Generated DSB Using the DR-GFP Reporter......Page 263
Analyzing Individual Repair Products......Page 264
Measuring SSA Using a Fluorescence Assay......Page 265
Determining the Frequency of V(D)J Recombination in a Chromosomal Substrate......Page 266
Assaying SSA of a RAG-Induced Chromosomal Break......Page 267
References......Page 268
Introduction......Page 304
Analysis of Rad55 Phosphorylation Status in Response to DNA Damage......Page 305
Reagents......Page 306
Procedure......Page 307
Comments to Protocol 1......Page 310
Vector System to Express Heterodimeric Proteins in S. cerevisiae......Page 311
Protocol 2: Purification of Rad55-Rad57 Heterodimers......Page 313
Procedure......Page 314
Protocol 3: Pull-Down of GST-Tagged Rad53 Kinase......Page 315
Procedure......Page 316
Comments to Protocol 3......Page 317
Procedure......Page 318
Acknowledgments......Page 319
References......Page 320
Introduction......Page 321
Mutator Assay......Page 322
Intrachromosomal Recombination Assay......Page 324
Checkpoint Responses to Replication Stalling and DNA Damage......Page 325
Assay of Cds1 Kinase Activity......Page 327
Assay of Chk1 Activation......Page 328
Chromatin Fractionation Assay......Page 329
Checkpoint Regulation of Mutagenesis......Page 330
References......Page 331
Introduction......Page 333
Terminology......Page 334
Mutant Accumulation......Page 335
Experimental Design......Page 337
Analyzing the Results of Fluctuation Assays......Page 340
Calculating the Mutation Rate......Page 345
Confidence Limits for p0......Page 346
Confidence Limits for m Obtained from the MSS Maximum Likelihood Method......Page 347
Phenotype Lag......Page 348
References......Page 349
Introduction......Page 352
Array-Based Screening......Page 353
Pinning Procedure......Page 355
Robotic Pinning Systems......Page 356
Barcode-Based Screening......Page 357
Protocol for Barcode-Based Chemical-Genetic Screening......Page 359
Synthetic Genetic Array Analysis to Identify DNA Damage Response Pathways......Page 361
Clustering Analysis......Page 362
How to Use Cluster 3.0 and Java Treeview......Page 364
Genome-Wide Screens for Suppressors of Genomic Instability......Page 367
Using SGA to Cross Reporters into Deletion Mutant Arrays......Page 369
Screening Using a Patch Assay......Page 370
Media Recipes......Page 371
References......Page 372
Sources of Antibodies to gamma-H2AX......Page 375
Immunocytochemical Detection of gamma-H2AX Foci in Mammalian Material......Page 376
Preparation of Tissue Touchprints......Page 378
Immunocytochemistry, Single Label......Page 379
Combined Immunocytochemistry and FISH on Metaphase Spreads......Page 380
Immunocytochemistry gamma-H2AX on Metaphase Spreads (FISH Compatible)......Page 381
Telomere FISH on Metaphase Spreads......Page 382
Immunocytochemical Detection of gamma-H2AX Foci in Budding Yeast......Page 383
Formation of DNA DSBs in Budding Yeast......Page 384
Visualization of gamma-foci in Budding Yeast......Page 385
Protein Extraction......Page 386
Chromatin Immunoprecipitation Using Yeast Anti-gamma-H2AX......Page 387
Further Reading......Page 389
Introduction......Page 390
Method......Page 391
Materials......Page 392
Materials......Page 393
Method......Page 394
IR-Induced Cell Cycle Checkpoints......Page 395
Materials......Page 396
Method......Page 397
Data Analysis......Page 398
Materials......Page 401
Method......Page 402
Data Analysis......Page 403
Introduction......Page 405
controls......Page 406
Method......Page 407
Material......Page 408
Method for Preparing Cells......Page 410
Method for Fixation, Slide Preparation, and Staining......Page 411
Statistical Concepts......Page 412
Data Analysis......Page 413
Immunofluorescence......Page 415
Controls......Page 416
Materials......Page 417
Method......Page 418
Materials......Page 420
Data Analysis......Page 421
References......Page 422
Introduction......Page 424
Experimental Outline......Page 425
Plaque Preparation and Testing......Page 426
Sample Collection During Incubation at 36deg......Page 427
Culture Preparation and Inoculation......Page 428
Determination of Cell Viability......Page 429
Buffers/Solutions Needed......Page 430
Cell Breakage......Page 431
DNA Elution......Page 432
Adding PCR Master Mix to DNA......Page 433
Analysis of Real-Time PCR Data and Concentration Adjustment......Page 435
Measuring ssDNA by QAOS......Page 437
Mixing Native and Boiled DNA......Page 438
References......Page 439
Introduction......Page 440
Chemistry......Page 441
Procedure......Page 444
Precautions......Page 446
Full Molecule Bisulfite Modification Assay......Page 447
Potassium Permanganate and Osmium Tetroxide Chemical Probing......Page 448
Procedure......Page 449
Conclusion......Page 452
References......Page 453
Detection and Structural Analysis of R-Loops......Page 456
Introduction......Page 457
Precautions......Page 458
Discussion......Page 460
Structural Analysis of the R-Loop with Native Sodium Bisulfite Probing and DNA Sequencing......Page 461
Discussion......Page 463
Procedure......Page 464
Discussion......Page 467
References......Page 468
Overview......Page 470
CORE Cassettes......Page 474
Transformation Protocol......Page 476
Colony PCR Reaction Conditions to Identify Desired Isolates......Page 477
Protocol for Transformation with Oligonucleotide......Page 478
Protocol for Targeting Oligonucleotides to a Region Containing a DSB......Page 480
Break-Mediated Delitto Perfetto with Oligonucleotides in Diploid Cells......Page 481
Generation of Chromosomal Rearrangements......Page 482
References......Page 485
Assays for Transcriptional Mutagenesis in Active Genes......Page 487
Introduction......Page 488
ssDNA Production......Page 490
Second Strand Synthesis......Page 491
Purification of Closed Circular DNA Molecules......Page 493
Preparation of Competent E. coli Cells and Transformation Conditions......Page 494
Luciferase Assay......Page 495
RNA Extraction and RT-PCR......Page 496
Sub-Cloning of RT-PCR Product......Page 497
Conclusions......Page 498
References......Page 499
Introduction......Page 500
S100 Extract......Page 502
Nuclear Extracts......Page 503
Plasmid Labeling and Supercoiling Assay: Nucleosome Assembly Associated with DNA Synthesis (Fig. 1)......Page 504
Histone Deposition on Immobilized DNA (Fig. 2)......Page 506
Controls......Page 509
Global Recruitment of Repair and Chromatin Assembly Factors to Damaged Chromatin (Fig. 3)......Page 510
Monitoring Chromatin Assembly Locally at Damage Sites (Fig. 4)......Page 511
Conclusion and Perspectives......Page 513
References......Page 514
Introduction......Page 517
ATPase Activity......Page 518
Example Protocols for DNA Dependent ATPase Assay......Page 519
DNA Translocation Monitoring-Triplex Displacement Assay......Page 520
Preparation of the Triplex Substrate......Page 521
Detection of Superhelical Torsion-Cruciform Extrusion Assay......Page 522
Protocol for Monitoring the Generation of Altered Superhelical Torsion by Cruciform Extrusion......Page 523
Protein......Page 524
DNA Design......Page 525
Preparation of the Protein:DNA Complex......Page 527
Trapping of Conformational States......Page 528
References......Page 529
Introduction......Page 531
Outline of ChIP-Chip Technique......Page 532
ChIP-Chip Protocol for the Analysis of Protein Binding Profile in S-Phase......Page 533
Extract Preparation......Page 534
Washes......Page 535
DNA Amplification......Page 536
Hybridization......Page 538
Discrimination Analysis......Page 539
Alternative Protocol for DNA Amplification (IVT Amplification Method)......Page 541
Materials......Page 542
Location Analyses of DNA Replication Proteins and Replicated Regions......Page 549
References......Page 552
Measurement of Chromosomal DNA Single-Strand Breaks and Replication Fork Progression Rates......Page 553
General Considerations......Page 554
Materials......Page 555
Protocol......Page 556
Data Acquisition and Analysis......Page 557
General Considerations......Page 558
Protocol......Page 559
Introduction and Overview......Page 560
General Considerations......Page 561
Protocol......Page 562
General Considerations......Page 563
Solutions......Page 564
Labeling and Spreading......Page 565
Immunostaining......Page 566
Image Analysis......Page 567
References......Page 568
Introduction......Page 569
General Considerations for Cell Culture and UV Irradiation......Page 571
UV Treatment and Recovery Assay......Page 572
Sample Preparation and Analysis of Recovery......Page 574
DNA Degradation at Arrested Replication Forks......Page 575
UV Treatment and Degradation Assay......Page 576
Sample Preparation and Analysis of Degradation......Page 577
Plasmid Replication Intermediates Observed by 2D N/N Gel Analysis......Page 578
UV Irradiation and DNA Isolation......Page 579
Two-Dimensional Agarose Gel Electrophoresis......Page 581
Acknowledgments......Page 583
References......Page 584
Introduction......Page 586
Analysis of Stalled and Collapsed Replication Forks Using 2D Gel Electrophoresis......Page 587
Materials and Solutions......Page 589
Supernatant......Page 590
Materials and Solutions......Page 591
Procedure......Page 592
First and Second Dimension Gel Electrophoresis and Southern Hybridization......Page 593
Branch Migration of Joint Molecules......Page 594
Preparation of In Vivo-Crosslinked DNA Samples for Electron Microscopy Analysis......Page 596
Purification of Replication Intermediates by BND Cellulose Column Chromatography......Page 597
Purification of Replication Intermediates by Size-Exclusion Column......Page 598
Cell Background......Page 599
DNA Labeling......Page 600
Materials and Solutions......Page 601
Materials and Solutions......Page 602
Procedure......Page 603
Procedure......Page 604
References......Page 605
Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae......Page 607
Introduction......Page 608
Selection of Yeast Cells with Gross-Chromosomal Rearrangements......Page 610
Determining the Rate of Accumulating Gross-Chromosomal Rearrangements......Page 611
Breakpoint Mapping......Page 613
Breakpoint Junction Amplification and Sequencing......Page 615
Chromosome V Rearrangement Types......Page 617
Conclusion......Page 619
References......Page 621
Introduction......Page 622
Cell Manipulations......Page 624
Southern Blot, Using Sponge Downward Transfer......Page 625
Interpretation......Page 626
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 628
Plasmid Replication in the Presence of Topoisomerase Inhibitors......Page 630
Examples of 2D Gels with Blocked Replication Forks......Page 631
In Vivo Processing of Blocked Replication Forks......Page 633
Closing Comments......Page 634
References......Page 636
Poly(ADP-ribose) Polymerase-1 Activation During DNA Damage and Repair......Page 639
Methods......Page 640
PARP-1 Purification Protocol from Insect Cells......Page 641
ECH Sepharose 4B-3AB Affinity Purification of PARP-1......Page 642
Detection of PARP-1 Activity in Activity Blots......Page 643
Materials......Page 644
Protocol (for One Plate)......Page 645
Base Excision Repair Assay......Page 646
Materials......Page 647
Procedure. Phosphorylation of oligonucleotides......Page 648
Procedure......Page 649
Repair Assay......Page 650
Purification of the DNA Product......Page 652
Solutions......Page 653
Poly(ADP-Ribose) Synthesis and XRCC1 Recruitment at Laser Induced DNA Strand Breaks......Page 654
References......Page 655
Introduction......Page 657
Tdp1......Page 658
3\'-Phosphotyrosine DNA......Page 659
3\'-(4-Nitro)Phenyl Phosphate DNA......Page 662
3\'-(4-Methylumbelliferone) Phosphate DNA......Page 663
Kinetic Analysis......Page 664
DNA Binding......Page 667
References......Page 669
Assaying Double-Strand Break Repair Pathway Choice in Mammalian Cells Using a Targeted Endonuclease or the RAG Reco.........Page 671
Measuring the Nature and Frequency of Repair of DNA Double-Strand Breaks Generated by the I-SceI Endonuclease......Page 672
Measuring the Nature and Frequency of Repair of DSBs Generated by the RAG Recombinase......Page 674
Site Loss PCR......Page 677
PCR-Southern Assay......Page 678
Measuring HR of a Chromosomal I-SceI DSB......Page 679
Measuring the Frequency of Imprecise NHEJ of an I-SceI-Generated DSB......Page 680
Assaying the Frequency of SSA of an I-SceI-Generated DSB Using the DR-GFP Reporter......Page 681
Analyzing Individual Repair Products......Page 682
Measuring SSA Using a Fluorescence Assay......Page 683
Determining the Frequency of V(D)J Recombination in a Chromosomal Substrate......Page 684
Assaying SSA of a RAG-Induced Chromosomal Break......Page 685
References......Page 686