Modrich DNA Repair, Part A

1. Auflage 2006
ISBN: 978-0-08-046344-5
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 408, 624 Seiten, Web PDF

Reihe: Methods in Enzymology

DNA Repair, Part A provides detailed coverage of modern methods for molecular analysis of enzymes and enzyme systems that function in the maintenance of genome integrity. Coverage areas include base excision repair, nucleotide excision repair, translesion DNA polymerases, mismatch repair, genetic recombination, and double strand break repair. - A laboratory standard for more than 40 years - Over 400 volumes strong - Also available on ScienceDirect - Part A of a 2-part series

Autoren/Hrsg.

Modrich, Paul

Inhaltsverzeichnis

1;Cover Page;1
2;Table of Contents;6
3;Contributors of Volume 408;10
4;Volumes in Series;14
5;Chapter 1: Assays for Determining Lesion Bypass Efficiency and Mutagenicity of Site-Specific DNA Lesions In Vivo;37
5.1;Introduction;37
5.2;Genome Construction;38
5.3;Genome Normalization;41
5.4;Lesion Bypass (CRAB) Assay;43
5.5;Lesion Mutagenesis (REAP) Assay;46
5.6;Conclusion;49
5.7;References;50
6;Chapter 2: Oxidative DNA Glycosylases: Recipes from Cloning to Characterization;51
6.1;Introduction;51
6.2;Purification of DNA Glycosylases;52
6.3;Preparation of Oligonucleotide Substrates;55
6.4;Activity Assays for Oxidative DNA Glycosylases;60
6.5;Acknowledgment;67
6.6;References;67
7;Chapter 3: Purification and Characterization of NEIL1 and NEIL2, Members of a Distinct Family of Mammalian DNA Glycosylases for Repair of Oxidized Bases;69
7.1;Endogenous and Induced DNA Damage;70
7.2;Repair of Oxidized Base Lesions in the Genome;70
7.3;Two Classes of Oxidized Base-Specific Glycosylases;71
7.4;Human Genome Database Analysis and Discovery of MutM/Nei-Like Proteins;72
7.5;Enzymatic Activity of NEIL1 and NEIL2 with Duplex or Bubble Oligonucleotide Substrates;75
7.6;Detection of DNA Glycosylase Activity of Recombinant NEILs by Trapping Analysis;80
7.7;Acknowledgments;82
7.8;References;82
8;Chapter 4: Analysis of Base Excision DNA Repair of the Oxidative Lesion 2-Deoxyribonolactone and the Formation of DNA-Protein Cross-Links;84
8.1;Introduction;85
8.2;Preparation of dL-DNA Substrates;86
8.3;Analysis of dL-mediated Spontaneous DNA-Protein Cross-Links;92
8.4;Analysis of dL-Specific BER;93
8.5;Acknowledgments;98
8.6;References;98
9;Chapter 5: Isolation and Analyses of MutY Homologs (MYH);100
9.1;Introduction;101
9.2;Isolation of MutY and MYH Proteins;104
9.3;Functional Assays of MutY and MYH;108
9.4;Protein Interaction Assays;111
9.5;Acknowledgments;112
9.6;References;112
10;Chapter 6: Use of Yeast for Detection of Endogenous Abasic Lesions, Their Source, and Their Repair;115
10.1;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
10.2;Endogenous AP Sites Cause Cell Death in the Absence of Apn1, Apn2, and Rad1-Rad10;119
10.3;Use of the apn1 apn2 rad1 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA;121
10.4;Use of the apn1 apn2 rad14 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA;124
10.5;Conclusions and Model;125
10.6;Acknowledgments;126
10.7;References;126
11;Chapter 7: Activities and Mechanism of DNA Polymerase beta;127
11.1;Introduction;128
11.2;Kinetic Characteristics of DNA Synthesis;129
11.3;Measurement of dRP Lyase Activity;134
11.4;Measurement of In Vitro BER;137
11.5;Reagents;140
11.6;Acknowledgment;141
11.7;References;141
12;Chapter 8: Direct Removal of Alkylation Damage from DNA by AlkB and Related DNA Dioxygenases;144
12.1;Purification of AlkB, ABH2, and ABH3;147
12.2;14C-Methylated Substrates;149
12.3;Assays;150
12.4;In Vivo Assay: Survival of Alkylated Single-Stranded DNA Bacteriophage in E. coli AlkB Mutants;153
12.5;References;155
13;Chapter 9: Purification and Characterization of DNA Photolyases;157
13.1;Introduction;157
13.2;Overexpression and Purification of Photolyases;164
13.3;Spectroscopic Properties and Characterization of Purified Photolyases;173
13.4;Assays for DNA Binding and Repair by Photolyase;178
13.5;Acknowledgments;187
13.6;References;187
14;Chapter 10: Genetic and In Vitro Assays of DNA Deamination;192
14.1;Introduction;193
14.2;Escherichia coli Assay of Deamination;193
14.3;In Vitro Assay of Deamination;199
14.4;Conclusion;204
14.5;Acknowledgments;205
14.6;References;205
15;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
15.1;Introduction;207
15.2;Purification of Recombinant Proteins;208
15.3;Damage-Specific DNA-Binding Assays;212
15.4;In Vitro NER Assays;219
15.5;UV-Induced Ubiquitylation Mediated by the UV-DDB-E3 Complex;221
15.6;References;223
16;Chapter 12: Purification and Characterization of Escherichia coli and Human Nucleotide Excision Repair Enzyme Systems;225
16.1;Introduction;225
16.2;Substrates for In Vitro Repair Assays;228
16.3;Enzymes for Repair Assays;233
16.4;In Vitro Repair Assays;239
16.5;Concluding Comments;246
16.6;Acknowledgment;246
16.7;References;246
17;Chapter 13: Assays for Transcription Elongation by RNA Polymerase II Using Oligo(dC)-Tailed Template with Single DNA Damage;250
17.1;Introduction;250
17.2;Construction of Closed-Circular Duplex DNA Substrates Containing a DNA Lesion at a Specific Site;251
17.3;Preparation of an Oligo(dc)-Tailed Template from Closed-Circular Duplex DNA Substrates;252
17.4;RNAPII Elongation Reaction Using Oligo(dC)-Tailed Template;253
17.5;Hot Labeling Reaction;253
17.6;Cold Labeling Reaction;255
17.7;Preparation of RNA Transcripts from RNAPII Transcription Elongation Assay Using Oligo(dC)-Tailed Template;256
17.8;Analysis of Lesion-Bypassed RNAPII Elongation Transcripts;256
17.9;Reverse-Transcription Reaction for First-Strand CDNA Synthesis and PCR;257
17.10;Analysis of 3' Ligation-Mediated RT-PCR;257
17.11;Acknowledgments;258
17.12;References;258
18;Chapter 14: In Vivo Assays for Transcription-Coupled Repair;259
18.1;Introduction;259
18.2;Important General Considerations;262
18.3;Southern Blot Method;264
18.4;Ligation-Mediated PCR Method;274
18.5;Acknowledgments;280
18.6;References;280
19;Chapter 15: TFIIH Enzymatic Activities in Transcription and Nucleotide Excision Repair;282
19.1;Introduction;283
19.2;Purification of Human TFIIH from Wild-Type and Patient Cell Lines;284
19.3;DNA Substrate Preparation Procedure;286
19.4;Enzymatic Assays;287
19.5;Acknowledgments;297
19.6;References;298
20;Chapter 16: An Assay for Studying Ubiquitylation of RNA Polymerase II and Other Proteins in Crude Yeast Extracts;300
20.1;Introduction;300
20.2;Whole Cell Extract Preparation;301
20.3;Preparation of myc-Tagged Ubiquitin;303
20.4;Purification of RNA Polymerase II;303
20.5;In Vitro Ubiquitylation;306
20.6;References;308
21;Chapter 17: Analysis of the Excision Step in Human DNA Mismatch Repair;309
21.1;Introduction;309
21.2;Methods;311
21.3;Discussion;319
21.4;Acknowledgments;319
21.5;References;319
22;Chapter 18: Characterization of the "Mismatch Repairosome" and Its Role in the Processing of Modified Nucleosides In Vitro;321
22.1;Introduction;321
22.2;Construction of Substrates Containing Insertion/Deletion Loops;322
22.3;Construction of G/T and G/C Mismatch Repair Substrates;330
22.4;Preparation of Nuclear Extracts;333
22.5;In Vitro Mismatch Repair Assays;333
22.6;DNA Affinity Purification;336
22.7;Conclusions;338
22.8;Acknowledgments;338
22.9;References;338
23;Chapter 19: Analysis of DNA Mismatch Repair in Cellular Response to DNA Damage;339
23.1;Introduction;340
23.2;In Vitro Biochemistry Studies;342
23.3;Studies at Cellular Level;344
23.4;Animal Models;349
23.5;Acknowledgments;351
23.6;References;352
24;Chapter 20: Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors;354
24.1;Introduction;354
24.2;Purification and Characterization of umuD Gene Products;358
24.3;Characterization of Escherichia coli Y Family Polymerases In Vitro and In Vivo;364
24.4;Conclusions;371
24.5;Acknowledgments;372
24.6;References;372
25;Chapter 21: Measuring the Fidelity of Translesion DNA Synthesis;377
25.1;Introduction;377
25.2;Experimental Outline of Translesion Synthesis Fidelity Assays;378
25.3;Materials;380
25.4;Experimental Procedures;381
25.5;Applications of the Method;389
25.6;Acknowledgments;390
25.7;References;390
26;Chapter 22: DNA Polymerases for Translesion DNA Synthesis: Enzyme Purification and Mouse Models for Studying Their Function;391
26.1;Introduction;391
26.2;Purification of Specialized DNA Polymerases as Tagged Proteins Following Expression of Cloned Fusion Genes;392
26.3;Mouse Strains Defective in Specialized DNA Polymerases;399
26.4;References;409
27;Chapter 23: Purification and Characterization of Escherichia coli DNA Polymerase V;414
27.1;Introduction;415
27.2;Assay Methods;416
27.3;Purification of DNA Polymerase V;417
27.4;Acknowledgments;424
27.5;References;424
28;Chapter 24: Yeast and Human Translesion DNA Synthesis Polymerases: Expression, Purification, and Biochemical Characterization;426
28.1;Introduction;427
28.2;Expression of GST-Tagged TLS Polymerases in Yeast;428
28.3;Protein Purification;431
28.4;DNA Synthesis Assays;435
28.5;Special Considerations for Individual TLS Polymerases;440
28.6;Acknowledgment;441
28.7;References;441
29;Chapter 25: Localization of Y-Family Polymerases and the DNA Polymerase Switch in Mammalian Cells;443
29.1;Introduction;443
29.2;Cellular Localization of Translesion Synthesis Polymerases;444
29.3;Ubiquitination of PCNA and Interaction with POLeta;449
29.4;References;451
30;Chapter 26: Repair of DNA Double Strand Breaks: In Vivo Biochemistry;452
30.1;Introduction;452
30.2;Methods;457
30.3;Media;458
30.4;Galactose Induction;458
30.5;DNA Extraction;459
30.6;Southern Analysis Using Native and Denaturing Gels;459
30.7;Analysis of Single-Stranded Tail Formation Using Slot Blots;460
30.8;Polymerase Chain Reaction Analysis;461
30.9;Chromatin Immunoprecipitation Procedure;461
30.10;Comments;463
30.11;References;464
31;Chapter 27: Assays for Nonhomologous End Joining in Extracts;466
31.1;Introduction;466
31.2;Preparation of Human Whole Cell Extracts That Support NHEJ;468
31.3;Gel-Based Assay for NHEJ of Compatible DNA Ends;470
31.4;Quantitative PCR Assay for NHEJ of Compatible or Noncompatible DNA Ends;472
31.5;Analysis of Processing Events;476
31.6;Concluding Remarks;480
31.7;Acknowledgments;480
31.8;References;480
32;Chapter 28: Purification and Assays of Saccharomyces cerevisiae Homologous Recombination Proteins;481
32.1;Introduction;481
32.2;Expression and Purification of Homologous Recombination Proteins;484
32.3;Biochemical Systems for Studying Homologous DNA Pairing and Strand Exchange;492
32.4;Acknowledgment;497
32.5;References;497
33;Chapter 29: Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy;499
33.1;Introduction;500
33.2;Fluorescence Recovery after Photobleaching;501
33.3;Experimental Approach;502
33.4;Conclusions;518
33.5;Acknowledgments;518
33.6;References;519
34;Chapter 30: Synthetic Junctions as Tools to Identify and Characterize Holliday Junction Resolvases;521
34.1;Introduction;522
34.2;Preparation of Synthetic Holliday Junctions;525
34.3;Holliday Junction Resolution Assays;530
34.4;Acknowledgments;536
34.5;References;536
35;Chapter 31: In Vitro Nonhomologous DNA End Joining System;538
35.1;Introduction;538
35.2;Procedures;539
35.3;General Remarks;542
35.4;Concluding Remarks;544
35.5;References;545
36;Chapter 32: RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes;547
36.1;Introduction;547
36.2;Expression Vectors;549
36.3;Protein Expression and Purification;551
36.4;Oligonucleotide Probe Preparation;556
36.5;In Vitro Cleavage Assays;557
36.6;Electrophoretic Mobility Shift Assays (EMSA);559
36.7;In-Gel Cleavage Assays;560
36.8;Discussion;561
36.9;Acknowledgments;562
36.10;References;562
37;Chapter 33: Purification and Biochemical Characterization of Ataxia-Telangiectasia Mutated and Mre11/Rad50/Nbs1;565
37.1;Introduction;565
37.2;Purification of Dimeric ATM;566
37.3;Purification of Monomeric ATM;569
37.4;Anti-Flag Affinity Chromatography;569
37.5;Purification of the MRN Complex;570
37.6;ATM Kinase Assay;572
37.7;References;574
38;Author Index;577
39;Subject Index;605