DNA Repair

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Introduction......Page 10
Genome Construction......Page 11
Protocol for Preparing ss-M13 DNA Vector Starting Material......Page 12
Protocol for Generating M13 Site-Specific Genomes......Page 13
Genome Normalization......Page 14
Protocol for Genome Normalization......Page 15
Lesion Bypass (CRAB) Assay......Page 16
Protocol for Lesion Bypass (CRAB) Assay......Page 18
Lesion Mutagenesis (REAP) Assay......Page 19
Conclusion......Page 22
References......Page 23
Introduction......Page 25
Purification of DNA Glycosylases......Page 26
Preparation of Oligonucleotide Substrates......Page 29
Activity Assays with Purified Recombinant Enzymes......Page 34
Determination of Kinetic Constants Using Purified Enzymes......Page 35
Schiff Base Assay......Page 38
Preparation and Activity of Bacterial Cell Extracts......Page 39
Preparation of Human Whole Cell Extracts......Page 40
References......Page 41
Purification and Characterization of NEIL1 and NEIL2, Members of a Distinct Family of Mammalian DNA Glycosylases for Repair.........Page 44
Repair of Oxidized Base Lesions in the Genome......Page 45
Two Classes of Oxidized Base-Specific Glycosylases......Page 46
Purification of NEIL1 from E. coli......Page 47
Purification of Recombinant NEIL2 from E. coli......Page 49
Oligonucleotide Substrates......Page 50
Incision Assay of DNA Glycosylases......Page 52
Unique activity of NEIL1 and NEIL2 with DNA Bubble Substrates......Page 53
Detection of DNA Glycosylase Activity of Recombinant NEILs by Trapping Analysis......Page 55
Perspective/Concluding Remarks......Page 56
References......Page 57
Analysis of Base Excision DNA Repair of the Oxidative Lesion 2-Deoxyribonolactone and the Formation of DNA-Protein Cross-Link......Page 60
Introduction......Page 61
Preparation of an Oligonucleotide DNA Substrate Containing a Site-Specific dL Residue......Page 62
Verification of a dL Site in DNA......Page 64
Preparation of a Plasmid DNA Substrate with a Defined dL Residue......Page 65
Analysis of dL-mediated Spontaneous DNA-Protein Cross-Links......Page 68
Analysis of dL-Specific BER......Page 69
Determination of dL-Specific BER Patch Size Distribution......Page 70
Determination of BER Mode in the Complete Repair of dL......Page 72
References......Page 74
Isolation and Analyses of MutY Homologs (MYH)......Page 77
Introduction......Page 78
MutY Protein Expression......Page 81
MutY Protein Purification......Page 82
Purification of Recombinant Eukaryotic MYH Proteins......Page 83
MutY Gel Mobility Shift Assay......Page 85
MutY Glycosylase and Trapping Assays......Page 86
Lac+ Reversion Assay......Page 87
GST Pull-Down Assay......Page 88
References......Page 89
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 92
Endogenous AP Sites Cause Cell Death in the Absence of Apn1, Apn2, and Rad1-Rad10......Page 96
Use of the apn1 apn2 rad1 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA......Page 98
Use of the apn1 apn2 rad14 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA......Page 101
Conclusions and Model......Page 102
References......Page 103
Activities and Mechanism of DNA Polymerase beta......Page 105
Introduction......Page 106
Kinetic Mechanism......Page 107
Kinetic Approaches......Page 108
General Considerations......Page 111
Removal of the 5'-dRP Backbone of an AP Site......Page 112
DNA Substrate for dRP Lyase Activity Determination......Page 113
Product Analysis......Page 114
General Considerations......Page 115
Assay for In Vitro BER Capacity......Page 116
BER Assay......Page 117
DNA and Enzymes......Page 118
References......Page 119
Direct Removal of Alkylation Damage from DNA by AlkB and Related DNA Dioxygenases......Page 122
Purification of AlkB, ABH2, and ABH3......Page 125
Purification of His-Tagged AlkB, ABH2, and ABH3 Proteins from E. coli......Page 126
14C-Methylated Substrates......Page 127
Standard Assay Using 14C-Alkylated Substrates......Page 128
Direct Reversion of a Methylated DNA Base to the Unmodified Form......Page 129
Assaying ABH2/ABH3 in Crude Cell Extracts......Page 130
In Vivo Assay: Survival of Alkylated Single-Stranded DNA Bacteriophage in E. coli AlkB Mutants......Page 131
Survival of Alkylated M13 Phage......Page 132
References......Page 133
Historical Perspective......Page 135
Photolyase/Cryptochrome Family......Page 136
Structure of Photolyases......Page 139
Reaction Mechanism......Page 140
Expression......Page 142
Blue Sepharose Chromatography......Page 143
Rapid Purification Methods......Page 144
Expression......Page 145
Phenyl Sepharose Chromatography......Page 146
DEAE Cellulose/Blue Sepharose Tandem Chromatography......Page 147
Deazaflavin Class Pyr<>Pyr Photolyase......Page 148
Yield, Purity, and Chromophore Composition......Page 149
Removal of MBP......Page 150
Absorbance Spectra......Page 151
Absorbance Spectra......Page 153
Determining Chromophore Stoichiometry......Page 154
Reconstituting Photolyase from Apoenzyme and Chromophores......Page 155
Nitrocellulose Filter-Binding Assay......Page 156
Electrophoretic Mobility Shift Assays (EMSA)......Page 157
DNA Footprinting......Page 159
Transformation Assay......Page 160
Absorption......Page 161
Restriction Site Restoration......Page 163
Enzyme-Sensitive Site Assay......Page 164
References......Page 165
Genetic and In Vitro Assays of DNA Deamination......Page 171
Escherichia coli Assay of Deamination......Page 172
Bacterial Strain Choice and Relevant Repair Pathways......Page 173
Electrotransformation of Competent E. coli......Page 174
Counting Viable Cells and Mutated Cells......Page 175
Determining the Sequence Context of Mutations......Page 177
Preparation of Deaminases for Use in the In Vitro Assay......Page 178
Extraction......Page 179
Oligonucleotides......Page 180
Purification of Oligonucleotides......Page 181
Use of TDG to Resolve a T:G or U:G Mismatch......Page 182
Conclusion......Page 183
References......Page 184
Introduction......Page 186
Purification of Recombinant Proteins......Page 187
Cell Culture, Virus Infection, and Extract Preparation......Page 188
Purification of HR23B and Centrin 2......Page 189
Purification of UV-DDB......Page 190
Damage-Specific DNA-Binding Assays......Page 191
Preparation of Single-Stranded Circular Phagemid DNA......Page 193
Preparation of Double-Stranded DNA Containing a Site-Specific Lesion......Page 194
Preparation of Radiolabeled DNA Fragments for EMSA......Page 195
Electrophoretic Mobility Shift Assay......Page 196
DNase I Footprinting Assay......Page 197
In Vitro NER Assays......Page 198
UV-Induced Ubiquitylation of XPC In Vivo......Page 200
In Vitro Ubiquitylation Assay......Page 201
References......Page 202
Introduction......Page 204
Circular Substrates......Page 207
Linear DNA Substrates......Page 208
Circular Substrates for Low-Resolution Resynthesis Assay......Page 211
Escherichia coli Repair Proteins......Page 212
UvrB......Page 213
Mammalian Cell Extracts......Page 214
Purification from Native Source......Page 215
XPA......Page 216
XPF.ERCC1......Page 217
Excision Assay......Page 218
Excision with Cell-Free Extracts......Page 222
Low-Resolution Repair Synthesis......Page 223
High-Resolution Repair Synthesis......Page 224
References......Page 225
Introduction......Page 229
Solutions and Materials......Page 230
Preparation of an Oligo(dc)-Tailed Template from Closed-Circular Duplex DNA Substrates......Page 231
Solutions and Materials......Page 232
Cold Labeling Reaction......Page 234
Analysis of Lesion-Bypassed RNAPII Elongation Transcripts (Fig. 1B)......Page 235
Analysis of 3' Ligation-Mediated RT-PCR (Fig. 1C)......Page 236
References......Page 237
Introduction......Page 239
Important General Considerations......Page 242
Cells......Page 244
Damage and Repair......Page 245
Genomic DNA Precipitation with NaI, SDS, and Isopropanol......Page 246
Centrifugation in Equilibrium Density Gradients......Page 247
Sample Preparation and Electrophoresis......Page 248
Transfer of DNA from Gel to Membrane......Page 249
Prehybridization......Page 250
Preparation of Template for Synthesis of RNA Probe......Page 252
Membrane Washes......Page 253
Irradiation of Cells......Page 254
Cleavage of DNA at CPD......Page 255
LMPCR Reaction......Page 256
Sequencing Gel Analysis of LMPCR Products......Page 257
References......Page 260
TFIIH Enzymatic Activities in Transcription and Nucleotide Excision Repair......Page 263
Introduction......Page 264
Whole Cell Extract (WCE)......Page 265
Chromatography......Page 266
Immobilization of Biotinylated DNA Fragment on Magnetic Beads......Page 267
Principle......Page 268
Analysis of Results......Page 269
Principle......Page 270
Principle......Page 271
Dual Incision Reaction with WCE......Page 272
Principle......Page 273
Analysis of Results......Page 275
Analysis of Results......Page 276
Acknowledgments......Page 278
References......Page 279
Introduction......Page 281
Whole Cell Extract Preparation......Page 282
Purification of RNA Polymerase II......Page 284
In Vitro Ubiquitylation......Page 287
References......Page 289
Introduction......Page 291
Isolation of MutSalpha, MutLalpha, and EXOI......Page 293
Mismatch-Provoked Excision......Page 297
References......Page 301
Introduction......Page 303
Construction of Substrates Containing Insertion/Deletion Loops......Page 304
Annealing......Page 305
Purification......Page 306
Coating of Magnetic Beads with the (+/-) Substrate DNA......Page 307
Production of Bacteriophage M13K07......Page 308
Production of Single‐Stranded DNA......Page 310
Annealing and Purification of 3' and 5' Substrates......Page 311
Experimental Strategy......Page 312
Primer Extension......Page 313
Cesium Chloride Gradient Purification......Page 314
Mismatch Repair Assays with G/T and G/C Substrates......Page 315
Mismatch Repair Assay with Insertion/Deletion Substrates......Page 317
DNA Affinity Purification......Page 318
References......Page 320
Analysis of DNA Mismatch Repair in Cellular Response to DNA Damage......Page 322
Introduction......Page 323
Purification of MutSalpha......Page 325
Construction of DNA Substrates Containing a Defined DNA Adduct......Page 326
Clonogenic Survival Analysis......Page 327
Apoptotic Analysis......Page 329
Flow Cytometry Analysis......Page 330
Paraffin Embedding and Sectioning......Page 332
Peroxidase Staining......Page 333
Acknowledgments......Page 334
References......Page 335
Introduction......Page 337
Expression and Purification of UmuD and UmuD'......Page 341
RecA/ssDNA Coprotease-Facilitated Cleavage of UmuD In Vitro and In Vivo......Page 343
Alkaline Cleavage of UmuD......Page 344
ClpXP Degradation Assay......Page 345
Using Intrinsic Tryptophan Fluorescence to Determine Binding Constants between UmuD Proteins and Their Interaction Partners......Page 346
Preparation of Native DinB......Page 347
DinB-Dependent Bypass of N2-dG Adducts In Vitro......Page 348
UV‐Induced Mutagenesis and Survival Attributed to umuDC......Page 349
Chemical‐Induced Mutagenesis and Survival Attributed to DinB and UmuC......Page 350
Assay for the Rate of Nascent DNA Synthesis by Thymine Incorporation......Page 352
Role of DinB in Damage-Independent DNA Replication Stalling......Page 353
Conclusions......Page 354
References......Page 355
Introduction......Page 360
Experimental Outline of Translesion Synthesis Fidelity Assays......Page 361
Enzymes and Reagents......Page 363
Lesion Bypass DNA Polymerase Assays......Page 364
Quantitation of Oligonucleotide Recovery......Page 367
Hybridization of Recovered Oligonucleotide to Gapped DNA......Page 369
Electroporation and Plating......Page 370
Mutant Frequency Data and Calculation of Error Rates......Page 371
Applications of the Method......Page 372
References......Page 373
Introduction......Page 375
Expression Constructs......Page 376
Expression in E. coli, Yeast, or SF9 Cells Infected with Baculovirus......Page 377
Expression in Baculovirus‐Infected SF9 Cells......Page 378
Expression in Mammalian Cells......Page 379
Purification by Affinity Chromatography......Page 380
Subsequent Purification Steps and Storage......Page 381
Testing Enzyme Purity and Activity......Page 382
Measuring Cellular Sensitivity to DNA-Damaging Agents......Page 383
Response to the T‐Cell‐Dependent Antigen NP‐CG......Page 389
Analysis of SH by Polymerase Chain Reaction......Page 390
PCR Reactions......Page 391
Skin Cancer in Mice Exposed to UV Radiation......Page 392
References......Page 393
Purification and Characterization of Escherichia coli DNA Polymerase V......Page 399
Introduction......Page 400
Procedure......Page 401
Expression Vectors and Strains......Page 402
Cell Lysis......Page 403
Pellet Branch......Page 405
Phosphocellulose Chromatography......Page 407
References......Page 409
Yeast and Human Translesion DNA Synthesis Polymerases: Expression, Purification, and Biochemical Characterization......Page 412
Introduction......Page 413
Expression Vector......Page 414
Cloning......Page 415
Growth of Yeast Cells and Induction of Protein Expression......Page 416
Preparation of Yeast Cell Extract......Page 417
Purification by Binding to Glutathione-Sepharose Affinity Column......Page 418
DNA Substrates......Page 421
DNA Polymerase Assays......Page 423
Efficiency and Fidelity Assays......Page 424
Determination of Kinetic Parameters......Page 425
Special Considerations for Individual TLS Polymerases......Page 426
References......Page 427
Introduction......Page 430
Methodology......Page 431
Fixation of Cells and Visualization of eGFP Autofluorescence......Page 432
Materials......Page 433
Methodology......Page 434
Microscopy......Page 435
Methodology......Page 436
Methodology......Page 437
References......Page 438
Introduction......Page 439
Strains......Page 444
Galactose Induction......Page 445
Southern Analysis Using Native and Denaturing Gels......Page 446
Analysis of Single-Stranded Tail Formation Using Slot Blots......Page 447
Cross-Linking Chromatin......Page 448
Chromatin Immunoprecipitation......Page 449
Harvesting Cells......Page 450
References......Page 451
Introduction......Page 453
Cell Lines......Page 455
Protocol for Preparing Extract......Page 456
Protocol for the Preparation of End-Labeled DNA Substrate......Page 457
Results......Page 458
Protocol for the In Vitro NHEJ Reaction (Fig. 3)......Page 459
Protocol for the Measurement of Joining Efficiency by Quantitative PCR......Page 462
Protocol for PCR Amplification and Sequencing of DNA Junctions......Page 463
Results......Page 465
References......Page 467
Introduction......Page 468
Expression and Purification of Homologous Recombination Proteins......Page 471
Rad51 Protein......Page 472
RPA Protein......Page 474
RAD52 Protein......Page 476
Rad54 Protein......Page 477
Biochemical Systems for Studying Homologous DNA Pairing and Strand Exchange......Page 479
Homologous DNA Pairing and Strand Exchange Reaction......Page 480
The D-Loop Assay to Study Homologous DNA Pairing......Page 482
References......Page 484
Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy......Page 487
Introduction......Page 488
Fluorescence Recovery after Photobleaching......Page 489
Cloning the cDNA of Interest......Page 490
Stable Cell Line Selection......Page 492
Isolating GFP‐Positive Cells......Page 493
Characterization of Cells Expressing the GFP Fusion Protein......Page 495
DNA Damage Induction Methods......Page 496
Quantitative Photobleaching Experiments......Page 497
FRAP to Study Mobility in the Nucleus......Page 498
Residence Time of Nuclear Proteins in DNA Damage-Induced Foci......Page 500
When Proteins Do Not Accumulate in Foci......Page 501
Low Fluorescence Levels......Page 504
Nucleoplasmic Mobility in the Presence of Abundant Foci......Page 505
Acknowledgments......Page 506
References......Page 507
Synthetic Junctions as Tools to Identify and Characterize Holliday Junction Resolvases......Page 510
Synthetic Holliday Junctions......Page 511
Structural Features and HJ Resolution......Page 512
Sequence Considerations......Page 514
Preparation Procedure......Page 515
Assay Procedure......Page 519
Denaturing PAGE and Analysis......Page 520
Native PAGE and Analysis......Page 522
Adaptation to Analyze Immobilized Proteins......Page 523
References......Page 525
Introduction......Page 527
DNA‐PKcs......Page 528
Artemis......Page 529
NHEJ Reaction......Page 530
General Remarks......Page 531
Concluding Remarks......Page 533
References......Page 534
Introduction......Page 536
Expression Vectors......Page 538
RAG Proteins......Page 540
HMGB1......Page 542
Oligonucleotide Probe Preparation......Page 545
In Vitro Cleavage Assays......Page 546
Electrophoretic Mobility Shift Assays (EMSA)......Page 548
In-Gel Cleavage Assays......Page 549
Discussion......Page 550
References......Page 551
Introduction......Page 554
Procedure......Page 555
Procedure......Page 556
Procedure......Page 557
Procedure......Page 558
Solutions and Materials......Page 559
Procedure......Page 560
ATM Kinase Assay......Page 561
Solutions and Materials......Page 562
References......Page 563