DNA Repair, Part A

Introduction......Page 32
Genome Construction......Page 33
Protocol for Preparing ss-M13 DNA Vector Starting Material......Page 34
Protocol for Generating M13 Site-Specific Genomes......Page 35
Genome Normalization......Page 36
Protocol for Genome Normalization......Page 37
Lesion Bypass (CRAB) Assay......Page 38
Protocol for Lesion Bypass (CRAB) Assay......Page 40
Lesion Mutagenesis (REAP) Assay......Page 41
Conclusion......Page 44
References......Page 45
Introduction......Page 47
Purification of DNA Glycosylases......Page 48
Preparation of Oligonucleotide Substrates......Page 51
Activity Assays with Purified Recombinant Enzymes......Page 56
Determination of Kinetic Constants Using Purified Enzymes......Page 57
Schiff Base Assay......Page 60
Preparation and Activity of Bacterial Cell Extracts......Page 61
Preparation of Human Whole Cell Extracts......Page 62
References......Page 63
Purification and Characterization of NEIL1 and NEIL2, Members of a Distinct Family of Mammalian DNA Glycosylases for Repair.........Page 66
Repair of Oxidized Base Lesions in the Genome......Page 67
Two Classes of Oxidized Base-Specific Glycosylases......Page 68
Purification of NEIL1 from E. coli......Page 69
Purification of Recombinant NEIL2 from E. coli......Page 71
Oligonucleotide Substrates......Page 72
Incision Assay of DNA Glycosylases......Page 74
Unique activity of NEIL1 and NEIL2 with DNA Bubble Substrates......Page 75
Detection of DNA Glycosylase Activity of Recombinant NEILs by Trapping Analysis......Page 77
Perspective/Concluding Remarks......Page 78
References......Page 79
Analysis of Base Excision DNA Repair of the Oxidative Lesion 2-Deoxyribonolactone and the Formation of DNA-Protein Cross-Link......Page 82
Introduction......Page 83
Preparation of an Oligonucleotide DNA Substrate Containing a Site-Specific dL Residue......Page 84
Verification of a dL Site in DNA......Page 86
Preparation of a Plasmid DNA Substrate with a Defined dL Residue......Page 87
Analysis of dL-mediated Spontaneous DNA-Protein Cross-Links......Page 90
Analysis of dL-Specific BER......Page 91
Determination of dL-Specific BER Patch Size Distribution......Page 92
Determination of BER Mode in the Complete Repair of dL......Page 94
References......Page 96
Isolation and Analyses of MutY Homologs (MYH)......Page 99
Introduction......Page 100
MutY Protein Expression......Page 103
MutY Protein Purification......Page 104
Purification of Recombinant Eukaryotic MYH Proteins......Page 105
MutY Gel Mobility Shift Assay......Page 107
MutY Glycosylase and Trapping Assays......Page 108
Lac+ Reversion Assay......Page 109
GST Pull-Down Assay......Page 110
References......Page 111
AP Sites in DNA and Related 3\'-Blocked and 5\'-Blocked Single Strand Breaks Form an Abundant and Deleterious Class of Endogenous DNA Damage......Page 114
Endogenous AP Sites Cause Cell Death in the Absence of Apn1, Apn2, and Rad1-Rad10......Page 118
Use of the apn1 apn2 rad1 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA......Page 120
Use of the apn1 apn2 rad14 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA......Page 123
Conclusions and Model......Page 124
References......Page 125
Activities and Mechanism of DNA Polymerase beta......Page 127
Introduction......Page 128
Kinetic Mechanism......Page 129
Kinetic Approaches......Page 130
General Considerations......Page 133
Removal of the 5\'-dRP Backbone of an AP Site......Page 134
DNA Substrate for dRP Lyase Activity Determination......Page 135
Product Analysis......Page 136
General Considerations......Page 137
Assay for In Vitro BER Capacity......Page 138
BER Assay......Page 139
DNA and Enzymes......Page 140
References......Page 141
Direct Removal of Alkylation Damage from DNA by AlkB and Related DNA Dioxygenases......Page 144
Purification of AlkB, ABH2, and ABH3......Page 147
Purification of His-Tagged AlkB, ABH2, and ABH3 Proteins from E. coli......Page 148
14C-Methylated Substrates......Page 149
Standard Assay Using 14C-Alkylated Substrates......Page 150
Direct Reversion of a Methylated DNA Base to the Unmodified Form......Page 151
Assaying ABH2/ABH3 in Crude Cell Extracts......Page 152
In Vivo Assay: Survival of Alkylated Single-Stranded DNA Bacteriophage in E. coli AlkB Mutants......Page 153
Survival of Alkylated M13 Phage......Page 154
References......Page 155
Historical Perspective......Page 157
Photolyase/Cryptochrome Family......Page 158
Structure of Photolyases......Page 161
Reaction Mechanism......Page 162
Expression......Page 164
Blue Sepharose Chromatography......Page 165
Rapid Purification Methods......Page 166
Expression......Page 167
Phenyl Sepharose Chromatography......Page 168
DEAE Cellulose/Blue Sepharose Tandem Chromatography......Page 169
Deazaflavin Class Pyr<>Pyr Photolyase......Page 170
Yield, Purity, and Chromophore Composition......Page 171
Removal of MBP......Page 172
Absorbance Spectra......Page 173
Absorbance Spectra......Page 175
Determining Chromophore Stoichiometry......Page 176
Reconstituting Photolyase from Apoenzyme and Chromophores......Page 177
Nitrocellulose Filter-Binding Assay......Page 178
Electrophoretic Mobility Shift Assays (EMSA)......Page 179
DNA Footprinting......Page 181
Transformation Assay......Page 182
Absorption......Page 183
Restriction Site Restoration......Page 185
Enzyme-Sensitive Site Assay......Page 186
References......Page 187
Genetic and In Vitro Assays of DNA Deamination......Page 193
Escherichia coli Assay of Deamination......Page 194
Bacterial Strain Choice and Relevant Repair Pathways......Page 195
Electrotransformation of Competent E. coli......Page 196
Counting Viable Cells and Mutated Cells......Page 197
Determining the Sequence Context of Mutations......Page 199
Preparation of Deaminases for Use in the In Vitro Assay......Page 200
Extraction......Page 201
Oligonucleotides......Page 202
Purification of Oligonucleotides......Page 203
Use of TDG to Resolve a T:G or U:G Mismatch......Page 204
Conclusion......Page 205
References......Page 206
Introduction......Page 208
Purification of Recombinant Proteins......Page 209
Cell Culture, Virus Infection, and Extract Preparation......Page 210
Purification of HR23B and Centrin 2......Page 211
Purification of UV-DDB......Page 212
Damage-Specific DNA-Binding Assays......Page 213
Preparation of Single-Stranded Circular Phagemid DNA......Page 215
Preparation of Double-Stranded DNA Containing a Site-Specific Lesion......Page 216
Preparation of Radiolabeled DNA Fragments for EMSA......Page 217
Electrophoretic Mobility Shift Assay......Page 218
DNase I Footprinting Assay......Page 219
In Vitro NER Assays......Page 220
UV-Induced Ubiquitylation of XPC In Vivo......Page 222
In Vitro Ubiquitylation Assay......Page 223
References......Page 224
Introduction......Page 226
Circular Substrates......Page 229
Linear DNA Substrates......Page 230
Circular Substrates for Low-Resolution Resynthesis Assay......Page 233
Escherichia coli Repair Proteins......Page 234
UvrB......Page 235
Mammalian Cell Extracts......Page 236
Purification from Native Source......Page 237
XPA......Page 238
XPF.ERCC1......Page 239
Excision Assay......Page 240
Excision with Cell-Free Extracts......Page 244
Low-Resolution Repair Synthesis......Page 245
High-Resolution Repair Synthesis......Page 246
References......Page 247
Introduction......Page 251
Solutions and Materials......Page 252
Preparation of an Oligo(dc)-Tailed Template from Closed-Circular Duplex DNA Substrates......Page 253
Solutions and Materials......Page 254
Cold Labeling Reaction......Page 256
Analysis of Lesion-Bypassed RNAPII Elongation Transcripts (Fig. 1B)......Page 257
Analysis of 3\' Ligation-Mediated RT-PCR (Fig. 1C)......Page 258
References......Page 259
Introduction......Page 261
Important General Considerations......Page 264
Cells......Page 266
Damage and Repair......Page 267
Genomic DNA Precipitation with NaI, SDS, and Isopropanol......Page 268
Centrifugation in Equilibrium Density Gradients......Page 269
Sample Preparation and Electrophoresis......Page 270
Transfer of DNA from Gel to Membrane......Page 271
Prehybridization......Page 272
Preparation of Template for Synthesis of RNA Probe......Page 274
Membrane Washes......Page 275
Irradiation of Cells......Page 276
Cleavage of DNA at CPD......Page 277
LMPCR Reaction......Page 278
Sequencing Gel Analysis of LMPCR Products......Page 279
References......Page 282
TFIIH Enzymatic Activities in Transcription and Nucleotide Excision Repair......Page 285
Introduction......Page 286
Whole Cell Extract (WCE)......Page 287
Chromatography......Page 288
Immobilization of Biotinylated DNA Fragment on Magnetic Beads......Page 289
Principle......Page 290
Analysis of Results......Page 291
Principle......Page 292
Principle......Page 293
Dual Incision Reaction with WCE......Page 294
Principle......Page 295
Analysis of Results......Page 297
Analysis of Results......Page 298
Acknowledgments......Page 300
References......Page 301
Introduction......Page 303
Whole Cell Extract Preparation......Page 304
Purification of RNA Polymerase II......Page 306
In Vitro Ubiquitylation......Page 309
References......Page 311
Introduction......Page 313
Isolation of MutSalpha, MutLalpha, and EXOI......Page 315
Mismatch-Provoked Excision......Page 319
References......Page 323
Introduction......Page 325
Construction of Substrates Containing Insertion/Deletion Loops......Page 326
Annealing......Page 327
Purification......Page 328
Coating of Magnetic Beads with the (+/-) Substrate DNA......Page 329
Production of Bacteriophage M13K07......Page 330
Production of Single‐Stranded DNA\0......Page 332
Annealing and Purification of 3\' and 5\' Substrates......Page 333
Experimental Strategy......Page 334
Primer Extension......Page 335
Cesium Chloride Gradient Purification......Page 336
Mismatch Repair Assays with G/T and G/C Substrates......Page 337
Mismatch Repair Assay with Insertion/Deletion Substrates......Page 339
DNA Affinity Purification......Page 340
References......Page 342
Analysis of DNA Mismatch Repair in Cellular Response to DNA Damage......Page 344
Introduction......Page 345
Purification of MutSalpha......Page 347
Construction of DNA Substrates Containing a Defined DNA Adduct......Page 348
Clonogenic Survival Analysis......Page 349
Apoptotic Analysis......Page 351
Flow Cytometry Analysis......Page 352
Paraffin Embedding and Sectioning......Page 354
Peroxidase Staining......Page 355
Acknowledgments......Page 356
References......Page 357
Introduction......Page 359
Expression and Purification of UmuD and UmuD\'......Page 363
RecA/ssDNA Coprotease-Facilitated Cleavage of UmuD In Vitro and In Vivo......Page 365
Alkaline Cleavage of UmuD......Page 366
ClpXP Degradation Assay......Page 367
Using Intrinsic Tryptophan Fluorescence to Determine Binding Constants between UmuD Proteins and Their Interaction Partners......Page 368
Preparation of Native DinB......Page 369
DinB-Dependent Bypass of N2-dG Adducts In Vitro......Page 370
UV‐Induced Mutagenesis and Survival Attributed to umuDC\0......Page 371
Chemical‐Induced Mutagenesis and Survival Attributed to DinB and UmuC\0......Page 372
Assay for the Rate of Nascent DNA Synthesis by Thymine Incorporation......Page 374
Role of DinB in Damage-Independent DNA Replication Stalling......Page 375
Conclusions......Page 376
References......Page 377
Introduction......Page 382
Experimental Outline of Translesion Synthesis Fidelity Assays......Page 383
Enzymes and Reagents......Page 385
Lesion Bypass DNA Polymerase Assays......Page 386
Quantitation of Oligonucleotide Recovery......Page 389
Hybridization of Recovered Oligonucleotide to Gapped DNA......Page 391
Electroporation and Plating......Page 392
Mutant Frequency Data and Calculation of Error Rates......Page 393
Applications of the Method......Page 394
References......Page 395
Introduction......Page 397
Expression Constructs......Page 398
Expression in E. coli, Yeast, or SF9 Cells Infected with Baculovirus......Page 399
Expression in Baculovirus‐Infected SF9 Cells\0......Page 400
Expression in Mammalian Cells......Page 401
Purification by Affinity Chromatography......Page 402
Subsequent Purification Steps and Storage......Page 403
Testing Enzyme Purity and Activity......Page 404
Measuring Cellular Sensitivity to DNA-Damaging Agents......Page 405
Response to the T‐Cell‐Dependent Antigen NP‐CG\0......Page 411
Analysis of SH by Polymerase Chain Reaction......Page 412
PCR Reactions......Page 413
Skin Cancer in Mice Exposed to UV Radiation......Page 414
References......Page 415
Purification and Characterization of Escherichia coli DNA Polymerase V......Page 421
Introduction......Page 422
Procedure......Page 423
Expression Vectors and Strains......Page 424
Cell Lysis......Page 425
Pellet Branch......Page 427
Phosphocellulose Chromatography......Page 429
References......Page 431
Yeast and Human Translesion DNA Synthesis Polymerases: Expression, Purification, and Biochemical Characterization......Page 434
Introduction......Page 435
Expression Vector......Page 436
Cloning......Page 437
Growth of Yeast Cells and Induction of Protein Expression......Page 438
Preparation of Yeast Cell Extract......Page 439
Purification by Binding to Glutathione-Sepharose Affinity Column......Page 440
DNA Substrates......Page 443
DNA Polymerase Assays......Page 445
Efficiency and Fidelity Assays......Page 446
Determination of Kinetic Parameters......Page 447
Special Considerations for Individual TLS Polymerases......Page 448
References......Page 449
Introduction......Page 452
Methodology......Page 453
Fixation of Cells and Visualization of eGFP Autofluorescence......Page 454
Materials......Page 455
Methodology......Page 456
Microscopy......Page 457
Methodology......Page 458
Methodology......Page 459
References......Page 460
Introduction......Page 461
Strains......Page 466
Galactose Induction......Page 467
Southern Analysis Using Native and Denaturing Gels......Page 468
Analysis of Single-Stranded Tail Formation Using Slot Blots......Page 469
Cross-Linking Chromatin......Page 470
Chromatin Immunoprecipitation......Page 471
Harvesting Cells......Page 472
References......Page 473
Introduction......Page 475
Cell Lines......Page 477
Protocol for Preparing Extract......Page 478
Protocol for the Preparation of End-Labeled DNA Substrate......Page 479
Results......Page 480
Protocol for the In Vitro NHEJ Reaction (Fig. 3)......Page 481
Protocol for the Measurement of Joining Efficiency by Quantitative PCR......Page 484
Protocol for PCR Amplification and Sequencing of DNA Junctions......Page 485
Results......Page 487
References......Page 489
Introduction......Page 490
Expression and Purification of Homologous Recombination Proteins......Page 493
Rad51 Protein......Page 494
RPA Protein......Page 496
RAD52 Protein......Page 498
Rad54 Protein......Page 499
Biochemical Systems for Studying Homologous DNA Pairing and Strand Exchange......Page 501
Homologous DNA Pairing and Strand Exchange Reaction......Page 502
The D-Loop Assay to Study Homologous DNA Pairing......Page 504
References......Page 506
Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy......Page 509
Introduction......Page 510
Fluorescence Recovery after Photobleaching......Page 511
Cloning the cDNA of Interest......Page 512
Stable Cell Line Selection......Page 514
Isolating GFP‐Positive Cells\0......Page 515
Characterization of Cells Expressing the GFP Fusion Protein......Page 517
DNA Damage Induction Methods......Page 518
Quantitative Photobleaching Experiments......Page 519
FRAP to Study Mobility in the Nucleus......Page 520
Residence Time of Nuclear Proteins in DNA Damage-Induced Foci......Page 522
When Proteins Do Not Accumulate in Foci......Page 523
Low Fluorescence Levels......Page 526
Nucleoplasmic Mobility in the Presence of Abundant Foci......Page 527
Acknowledgments......Page 528
References......Page 529
Synthetic Junctions as Tools to Identify and Characterize Holliday Junction Resolvases......Page 532
Synthetic Holliday Junctions......Page 533
Structural Features and HJ Resolution......Page 534
Sequence Considerations......Page 536
Preparation Procedure......Page 537
Assay Procedure......Page 541
Denaturing PAGE and Analysis......Page 542
Native PAGE and Analysis......Page 544
Adaptation to Analyze Immobilized Proteins......Page 545
References......Page 547
Introduction......Page 549
DNA‐PKcs\0......Page 550
Artemis......Page 551
NHEJ Reaction......Page 552
General Remarks......Page 553
Concluding Remarks......Page 555
References......Page 556
Introduction......Page 558
Expression Vectors......Page 560
RAG Proteins......Page 562
HMGB1......Page 564
Oligonucleotide Probe Preparation......Page 567
In Vitro Cleavage Assays......Page 568
Electrophoretic Mobility Shift Assays (EMSA)......Page 570
In-Gel Cleavage Assays......Page 571
Discussion......Page 572
References......Page 573
Introduction......Page 576
Procedure......Page 577
Procedure......Page 578
Procedure......Page 579
Procedure......Page 580
Solutions and Materials......Page 581
Procedure......Page 582
ATM Kinase Assay......Page 583
Solutions and Materials......Page 584
References......Page 585