DNA Repair, Part A (eBook)

Cover Page 1
Table of Contents 6
Contributors of Volume 408 10
Volumes in Series 14
Chapter 1: Assays for Determining Lesion Bypass Efficiency and Mutagenicity of Site-Specific DNA Lesions In Vivo 37
Introduction 37
Genome Construction 38
Genome Normalization 41
Lesion Bypass (CRAB) Assay 43
Lesion Mutagenesis (REAP) Assay 46
Conclusion 49
References 50
Chapter 2: Oxidative DNA Glycosylases: Recipes from Cloning to Characterization 51
Introduction 51
Purification of DNA Glycosylases 52
Preparation of Oligonucleotide Substrates 55
Activity Assays for Oxidative DNA Glycosylases 60
Acknowledgment 67
References 67
Chapter 3: Purification and Characterization of NEIL1 and NEIL2, Members of a Distinct Family of Mammalian DNA Glycosylases for Repair of Oxidized Bases 69
Endogenous and Induced DNA Damage 70
Repair of Oxidized Base Lesions in the Genome 70
Two Classes of Oxidized Base-Specific Glycosylases 71
Human Genome Database Analysis and Discovery of MutM/Nei-Like Proteins 72
Enzymatic Activity of NEIL1 and NEIL2 with Duplex or Bubble Oligonucleotide Substrates 75
Detection of DNA Glycosylase Activity of Recombinant NEILs by Trapping Analysis 80
Acknowledgments 82
References 82
Chapter 4: Analysis of Base Excision DNA Repair of the Oxidative Lesion 2-Deoxyribonolactone and the Formation of DNA-Protein Cross-Links 84
Introduction 85
Preparation of dL-DNA Substrates 86
Analysis of dL-mediated Spontaneous DNA-Protein Cross-Links 92
Analysis of dL-Specific BER 93
Acknowledgments 98
References 98
Chapter 5: Isolation and Analyses of MutY Homologs (MYH) 100
Introduction 101
Isolation of MutY and MYH Proteins 104
Functional Assays of MutY and MYH 108
Protein Interaction Assays 111
Acknowledgments 112
References 112
Chapter 6: Use of Yeast for Detection of Endogenous Abasic Lesions, Their Source, and Their Repair 115
AP Sites in DNA and Related 3'-Blocked and 5'-Blocked Single Strand Breaks Form an Abundant and Deleterious Class of Endogenous DNA Damage 115
Endogenous AP Sites Cause Cell Death in the Absence of Apn1, Apn2, and Rad1-Rad10 119
Use of the apn1 apn2 rad1 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA 121
Use of the apn1 apn2 rad14 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA 124
Conclusions and Model 125
Acknowledgments 126
References 126
Chapter 7: Activities and Mechanism of DNA Polymerase beta 127
Introduction 128
Kinetic Characteristics of DNA Synthesis 129
Measurement of dRP Lyase Activity 134
Measurement of In Vitro BER 137
Reagents 140
Acknowledgment 141
References 141
Chapter 8: Direct Removal of Alkylation Damage from DNA by AlkB and Related DNA Dioxygenases 144
Purification of AlkB, ABH2, and ABH3 147
14C-Methylated Substrates 149
Assays 150
In Vivo Assay: Survival of Alkylated Single-Stranded DNA Bacteriophage in E. coli AlkB Mutants 153
References 155
Chapter 9: Purification and Characterization of DNA Photolyases 157
Introduction 157
Overexpression and Purification of Photolyases 164
Spectroscopic Properties and Characterization of Purified Photolyases 173
Assays for DNA Binding and Repair by Photolyase 178
Acknowledgments 187
References 187
Chapter 10: Genetic and In Vitro Assays of DNA Deamination 192
Introduction 193
Escherichia coli Assay of Deamination 193
In Vitro Assay of Deamination 199
Conclusion 204
Acknowledgments 205
References 205
Chapter 11: The Xeroderma Pigmentosum Group C Protein Complex and Ultraviolet-Damaged DNA-Binding Protein: Functional Assays for Damage Recognition Factors Involved in Global Genome Repair 207
Introduction 207
Purification of Recombinant Proteins 208
Damage-Specific DNA-Binding Assays 212
In Vitro NER Assays 219
UV-Induced Ubiquitylation Mediated by the UV-DDB-E3 Complex 221
References 223
Chapter 12: Purification and Characterization of Escherichia coli and Human Nucleotide Excision Repair Enzyme Systems 225
Introduction 225
Substrates for In Vitro Repair Assays 228
Enzymes for Repair Assays 233
In Vitro Repair Assays 239
Concluding Comments 246
Acknowledgment 246
References 246
Chapter 13: Assays for Transcription Elongation by RNA Polymerase II Using Oligo(dC)-Tailed Template with Single DNA Damage 250
Introduction 250
Construction of Closed-Circular Duplex DNA Substrates Containing a DNA Lesion at a Specific Site 251
Preparation of an Oligo(dc)-Tailed Template from Closed-Circular Duplex DNA Substrates 252
RNAPII Elongation Reaction Using Oligo(dC)-Tailed Template 253
Hot Labeling Reaction 253
Cold Labeling Reaction 255
Preparation of RNA Transcripts from RNAPII Transcription Elongation Assay Using Oligo(dC)-Tailed Template 256
Analysis of Lesion-Bypassed RNAPII Elongation Transcripts 256
Reverse-Transcription Reaction for First-Strand CDNA Synthesis and PCR 257
Analysis of 3' Ligation-Mediated RT-PCR 257
Acknowledgments 258
References 258
Chapter 14: In Vivo Assays for Transcription-Coupled Repair 259
Introduction 259
Important General Considerations 262
Southern Blot Method 264
Ligation-Mediated PCR Method 274
Acknowledgments 280
References 280
Chapter 15: TFIIH Enzymatic Activities in Transcription and Nucleotide Excision Repair 282
Introduction 283
Purification of Human TFIIH from Wild-Type and Patient Cell Lines 284
DNA Substrate Preparation Procedure 286
Enzymatic Assays 287
Acknowledgments 297
References 298
Chapter 16: An Assay for Studying Ubiquitylation of RNA Polymerase II and Other Proteins in Crude Yeast Extracts 300
Introduction 300
Whole Cell Extract Preparation 301
Preparation of myc-Tagged Ubiquitin 303
Purification of RNA Polymerase II 303
In Vitro Ubiquitylation 306
References 308
Chapter 17: Analysis of the Excision Step in Human DNA Mismatch Repair 309
Introduction 309
Methods 311
Discussion 319
Acknowledgments 319
References 319
Chapter 18: Characterization of the "Mismatch Repairosome" and Its Role in the Processing of Modified Nucleosides In Vitro 321
Introduction 321
Construction of Substrates Containing Insertion/Deletion Loops 322
Construction of G/T and G/C Mismatch Repair Substrates 330
Preparation of Nuclear Extracts 333
In Vitro Mismatch Repair Assays 333
DNA Affinity Purification 336
Conclusions 338
Acknowledgments 338
References 338
Chapter 19: Analysis of DNA Mismatch Repair in Cellular Response to DNA Damage 339
Introduction 340
In Vitro Biochemistry Studies 342
Studies at Cellular Level 344
Animal Models 349
Acknowledgments 351
References 352
Chapter 20: Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors 354
Introduction 354
Purification and Characterization of umuD Gene Products 358
Characterization of Escherichia coli Y Family Polymerases In Vitro and In Vivo 364
Conclusions 371
Acknowledgments 372
References 372
Chapter 21: Measuring the Fidelity of Translesion DNA Synthesis 377
Introduction 377
Experimental Outline of Translesion Synthesis Fidelity Assays 378
Materials 380
Experimental Procedures 381
Applications of the Method 389
Acknowledgments 390
References 390
Chapter 22: DNA Polymerases for Translesion DNA Synthesis: Enzyme Purification and Mouse Models for Studying Their Function 391
Introduction 391
Purification of Specialized DNA Polymerases as Tagged Proteins Following Expression of Cloned Fusion Genes 392
Mouse Strains Defective in Specialized DNA Polymerases 399
References 409
Chapter 23: Purification and Characterization of Escherichia coli DNA Polymerase V 414
Introduction 415
Assay Methods 416
Purification of DNA Polymerase V 417
Acknowledgments 424
References 424
Chapter 24: Yeast and Human Translesion DNA Synthesis Polymerases: Expression, Purification, and Biochemical Characterization 426
Introduction 427
Expression of GST-Tagged TLS Polymerases in Yeast 428
Protein Purification 431
DNA Synthesis Assays 435
Special Considerations for Individual TLS Polymerases 440
Acknowledgment 441
References 441
Chapter 25: Localization of Y-Family Polymerases and the DNA Polymerase Switch in Mammalian Cells 443
Introduction 443
Cellular Localization of Translesion Synthesis Polymerases 444
Ubiquitination of PCNA and Interaction with POLeta 449
References 451
Chapter 26: Repair of DNA Double Strand Breaks: In Vivo Biochemistry 452
Introduction 452
Methods 457
Media 458
Galactose Induction 458
DNA Extraction 459
Southern Analysis Using Native and Denaturing Gels 459
Analysis of Single-Stranded Tail Formation Using Slot Blots 460
Polymerase Chain Reaction Analysis 461
Chromatin Immunoprecipitation Procedure 461
Comments 463
References 464
Chapter 27: Assays for Nonhomologous End Joining in Extracts 466
Introduction 466
Preparation of Human Whole Cell Extracts That Support NHEJ 468
Gel-Based Assay for NHEJ of Compatible DNA Ends 470
Quantitative PCR Assay for NHEJ of Compatible or Noncompatible DNA Ends 472
Analysis of Processing Events 476
Concluding Remarks 480
Acknowledgments 480
References 480
Chapter 28: Purification and Assays of Saccharomyces cerevisiae Homologous Recombination Proteins 481
Introduction 481
Expression and Purification of Homologous Recombination Proteins 484
Biochemical Systems for Studying Homologous DNA Pairing and Strand Exchange 492
Acknowledgment 497
References 497
Chapter 29: Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy 499
Introduction 500
Fluorescence Recovery after Photobleaching 501
Experimental Approach 502
Conclusions 518
Acknowledgments 518
References 519
Chapter 30: Synthetic Junctions as Tools to Identify and Characterize Holliday Junction Resolvases 521
Introduction 522
Preparation of Synthetic Holliday Junctions 525
Holliday Junction Resolution Assays 530
Acknowledgments 536
References 536
Chapter 31: In Vitro Nonhomologous DNA End Joining System 538
Introduction 538
Procedures 539
General Remarks 542
Concluding Remarks 544
References 545
Chapter 32: RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes 547
Introduction 547
Expression Vectors 549
Protein Expression and Purification 551
Oligonucleotide Probe Preparation 556
In Vitro Cleavage Assays 557
Electrophoretic Mobility Shift Assays (EMSA) 559
In-Gel Cleavage Assays 560
Discussion 561
Acknowledgments 562
References 562
Chapter 33: Purification and Biochemical Characterization of Ataxia-Telangiectasia Mutated and Mre11/Rad50/Nbs1 565
Introduction 565
Purification of Dimeric ATM 566
Purification of Monomeric ATM 569
Anti-Flag Affinity Chromatography 569
Purification of the MRN Complex 570
ATM Kinase Assay 572
References 574
Author Index 577
Subject Index 605