Modrich DNA Repair, Part B

1. Auflage 2011
ISBN: 978-0-08-046467-1
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 409, 616 Seiten, Web PDF

Reihe: Methods in Enzymology

This volume emphasizes the intracellular consequences of DNA damage, describing procedures for analysis of checkpoint responses, DNA repair in vivo, replication fork encounter of DNA damage, as well as biological methods for analysis of mutation production and chromosome rearrangements. It also describes molecular methods for analysis of a number of genome maintenance activities including DNA ligases, helicases, and single-strand binding proteins.*Part B of a 2-part series*Addresses DNA maintenance enzymes*Discusses damage signaling*Presents In vivo analysis of DNA repair*Covers mutation and chromosome rearrangements

Autoren/Hrsg.

Modrich, Paul

Inhaltsverzeichnis

1;Cover Page;1
2;Table of Contents;6
3;Contributors to Volume 409;10
4;Volume in Series;16
5;Chapter 1: Overproduction and Purification of RFC-Related Clamp Loaders and PCNA-Related Clamps from Saccharomyces cerevisiae;39
5.1;Introduction;39
5.2;Overproduction of Complexes in Yeast;41
5.3;Extract Preparation and Purification;43
5.4;Discussion;47
5.5;Acknowledgments;48
5.6;References;48
6;Chapter 2: Functional Assays for Replication Protein A (RPA);49
6.1;Introduction;50
6.2;Purification of Recombinant RPA;51
6.3;DNA-Binding Assays;54
6.4;Gel-Binding Assays (Gel Mobility Shift and Helix-Destabilization);54
6.5;Surface Plasmon Resonance;59
6.6;Fluorescence Polarization;64
6.7;Other DNA-Binding Assays;65
6.8;RPA-Protein Interactions (Enzyme-Linked Immunosorbent Assay);65
6.9;Functional Assays for RPA;67
6.10;In Vivo Localization;69
6.11;In Vivo Localization by Indirect Immunofluorescence;69
6.12;Summary and Conclusion;72
6.13;References;72
7;Chapter 3: Human DNA Ligases I, III, and IV-Purification and New Specific Assays for These Enzymes;77
7.1;Introduction;77
7.2;Overexpression and Purification of Human DNA Ligases;78
7.3;DNA Ligation Assays;82
7.4;Biotinylated Linear DNA Substrates with Blocked DNA Ends;83
7.5;Linear DNA Substrates to Measure Intra- and Intermolecular DNA Joining;84
7.6;Ligation Assays Using Fluorescent DNA Substrates;85
7.7;Concluding Remarks;87
7.8;Acknowledgments;88
7.9;References;88
8;Chapter 4: Enzymatic Mechanism of the WRN Helicase/Nuclease;90
8.1;Expression and Purification of Recombinant WRN Protein;91
8.2;Characterization of WRN Helicase Activity;92
8.3;Helicase Assays;95
8.4;Preparation of Radiolabeled Duplex DNA Substrate;96
8.5;Preparation of Four-Stranded Oligonucleotide-Based Holliday Junction;97
8.6;Preparation of Three-Stranded Oligonucleotide-Based D-Loop;98
8.7;Radiometric Helicase Assay;100
8.8;Real-Time Fluorometric Helicase Assay;102
8.9;ATPase Assay;105
8.10;Gel Mobility Shift Assay to Measure DNA Binding;107
8.11;Exonuclease Assay;109
8.12;Effects of Post-Translational Modifications on WRN Catalytic Activities;116
8.13;WRN Protein Interactions that Modulate WRN Catalytic Activity;118
8.14;Acknowledgments;120
8.15;References;120
9;Chapter 5: Analysis of the DNA Unwinding Activity of RecQ Family Helicases;124
9.1;Introduction;124
9.2;Preparation of Substrates;125
9.3;Analysis of Helicase Activity;132
9.4;Safety Issues;136
9.5;References;137
10;Chapter 6: Characterization of Checkpoint Responses to DNA Damage in Saccharomyces cerevisiae: Basic Protocols;139
10.1;General Introduction;139
10.2;Budding Analysis;140
10.3;Microcolony Assay;143
10.4;Visualization of the Mitotic Spindle;145
10.5;Nuclear Staining with DAPI;148
10.6;Flow Cytometry;149
10.7;Analysis of Rad53 Phosphorylation;152
10.8;Acknowledgments;154
10.9;References;154
11;Chapter 7: Recruitment of ATR-ATRIP, Rad17, and 9-1-1 Complexes to DNA Damage;156
11.1;Introduction;156
11.2;Chromatin Association of ATR, Rad17, Rad9, and RPA;158
11.3;Purification of ATR, ATRIP, and Reconstitution of the ATR-ATRIP Complex;160
11.4;Recruitment of ATRIP and ATR-ATRIP to RPA-Coated Single-Stranded DNA;161
11.5;Purification of Rad17 and 9-1-1 Complexes;163
11.6;Recruitment of 9-1-1 Complexes to Primed ssDNA by RPA and Rad17 Complexes;164
11.7;Conclusion;166
11.8;Acknowledgments;167
11.9;References;167
12;Chapter 8: Multiple Approaches to Study S. cerevisiae Rad9, a Prototypical Checkpoint Protein;169
12.1;Introduction;169
12.2;Cell Cycle Checkpoint Analysis;171
12.3;Summary;185
12.4;Acknowledgments;186
12.5;References;186
13;Chapter 9: Methods for Studying Adaptation to the DNA Damage Checkpoint in Yeast;188
13.1;Introduction;188
13.2;Designing Adaptation Assays;189
13.3;Ensuring that DSBs Are Irreparable;190
13.4;IR-Induced DSBs;191
13.5;HO-Induced DSBs;192
13.6;Using Disomic Strains;194
13.7;Adaptation Assays Based on Chromosome Loss;199
13.8;Adaptation to cdc13-Induced Damage;201
13.9;Conclusions;202
13.10;References;203
14;Chapter 10: DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae;204
14.1;Introduction;204
14.2;Methods;205
14.3;Purification of Rad55-Rad57 Heterodimer from S. cerevisiae;213
14.4;In Vitro Kinase Assays;215
14.5;Conclusions and Perspectives;219
14.6;Acknowledgments;219
14.7;References;220
15;Chapter 11: Methods for Studying Mutagenesis and Checkpoints in Schizosaccharomyces pombe;221
15.1;Introduction;221
15.2;Protocol;222
15.3;Protocol;227
15.4;References;231
16;Chapter 12: Methods for Determining Spontaneous Mutation Rates;233
16.1;Introduction;233
16.2;Terminology;234
16.3;The Lea-Coulson Model;235
16.4;Mutant Accumulation;235
16.5;Fluctuation Analysis;237
16.6;Acknowledgments;249
16.7;References;249
17;Chapter 13: Genomic Approaches for Identifying DNA Damage Response Pathways in S. cerevisiae;251
17.1;Introduction;251
17.2;Systematic Genome-Wide Screens to Identify Genes Required for Resistance to DNA Damaging Agents;252
17.3;Synthetic Genetic Array Analysis to Identify DNA Damage Response Pathways;260
17.4;Acknowledgments;271
17.5;References;271
18;Chapter 14: Techniques for gamma-H2AX Detection;274
18.1;Introduction;274
18.2;Sources of Antibodies to gamma-H2AX;274
18.3;Immunocytochemical Detection of gamma-H2AX Foci in Mammalian Material;275
18.4;Combined Immunocytochemistry and FISH on Metaphase Spreads;279
18.5;Immunocytochemical Detection of gamma-H2AX Foci in Budding Yeast;282
18.6;Immunoblot Detection of Yeast gamma-H2A;285
18.7;Chromatin Immunoprecipitation Using Yeast Anti-gamma-H2AX;286
18.8;References;288
18.9;Further Reading;288
19;Chapter 15: Methods for Studying the Cellular Response to DNA Damage: Influence of the Mre11 Complex on Chromosome Metabolism;289
19.1;Introduction;289
19.2;Derivation of Primary and Transformed Cells;290
19.3;IR-Induced Cell Cycle Checkpoints;294
19.4;Metaphase Spreads;307
19.5;Immunofluorescence;314
19.6;Acknowledgments;321
19.7;References;321
20;Chapter 16: Detecting Repair Intermediates In Vivo: Effects of DNA Damage Response Genes on Single-Stranded DNA Accumulation at Uncapped Telomeres in Budding Yeast;323
20.1;Introduction;323
20.2;Experimental Outline;324
20.3;Methods;325
20.4;Acknowledgments;338
20.5;References;338
21;Chapter 17: Analysis of Non-B DNA Structure at Chromosomal Sites in the Mammalian Genome;339
21.1;Introduction;339
21.2;Specific Methods;340
21.3;Potassium Permanganate and Osmium Tetroxide Chemical Probing;347
21.4;Conclusion;351
21.5;Acknowledgments;352
21.6;References;352
22;Chapter 18: Detection and Structural Analysis of R-Loops;354
22.1;Introduction;355
22.2;Procedures;356
22.3;References;366
23;Chapter 19: The Delitto Perfetto Approach to In Vivo Site-Directed Mutagenesis and Chromosome Rearrangements with Synthetic Oligonucleotides in Yeast;367
23.1;Overview;367
23.2;Creation and Utility of Delitto Perfetto Systems;371
23.3;Conclusions;382
23.4;References;382
24;Chapter 20: Assays for Transcriptional Mutagenesis in Active Genes;383
24.1;Introduction;384
24.2;ssDNA Production;386
24.3;Synthesis of the Transcribed Strand;387
24.4;Purification of Closed Circular DNA Molecules;389
24.5;Quantification of Transcriptional Mutagenesis;390
24.6;Determining the Transcriptional Mutagenesis Spectrum;392
24.7;Conclusions;394
24.8;Acknowledgments;395
24.9;References;395
25;Chapter 21: Methods for Studying Chromatin Assembly Coupled to DNA Repair;396
25.1;Introduction;396
25.2;Analysis of Chromatin Assembly Coupled to DNA Repair In Vitro;398
25.3;Analysis of Chromatin Assembly Coupled to DNA Repair In Vivo;406
25.4;Conclusion and Perspectives;409
25.5;Acknowledgments;410
25.6;References;410
26;Chapter 22: Structure-Function Analysis of SWI2/SNF2 Enzymes;413
26.1;Introduction;413
26.2;Biochemical Characterization of SWI2/SNF2 ATPases;414
26.3;DNA Translocation Monitoring-Triplex Displacement Assay;416
26.4;Detection of Superhelical Torsion-Cruciform Extrusion Assay;418
26.5;Crystallization of Protein:DNA Complexes;420
26.6;Conclusions;425
26.7;Acknowledgments;425
26.8;References;425
27;Chapter 23: Genomic Approach for the Understanding of Dynamic Aspect of Chromosome Behavior;427
27.1;Introduction;427
27.2;Outline of ChIP-Chip Technique;428
27.3;ChIP-Chip Protocol for the Analysis of Protein Binding Profile in S-Phase;429
27.4;Solutions for ChIP-chip Analysis;430
27.5;Location Analyses of DNA Replication Proteins and Replicated Regions;445
27.6;Acknowledgments;448
27.7;Reference;448
28;Chapter 24: Measurement of Chromosomal DNA Single-Strand Breaks and Replication Fork Progression Rates;448
28.1;Measurement of Chromosomal Single-Strand Breaks by Alkaline Single-Cell Agarose-Gel Electrophoresis (Alkaline Comet Assay);449
28.2;Identification of Novel SSBR Polypeptides by Yeast 2-Hybrid Library Screens;453
28.3;Recovery of XRCC1 Protein Complexes from Cell Extract by Immobilized Metal-Chelate Chromatography (IMAC);455
28.4;Quantification of Chromosomal Replication Fork Rates on Damaged DNA by DNA Fiber Labeling;458
28.5;References;463
29;Chapter 25: Monitoring DNA Replication Following UV-Induced Damage in Escherichia coli;463
29.1;Introduction;463
29.2;Description of the Methods and Technical Comments;465
29.3;Concluding Remarks;477
29.4;Acknowledgments;477
29.5;References;478
30;Chapter 26: Methods to Study Replication Fork Collapse in Budding Yeast;480
30.1;Introduction;480
30.2;Methods;481
30.3;Cell Background;493
30.4;Acknowledgments;499
30.5;References;499
31;Chapter 27: Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae;500
31.1;Introduction;501
31.2;Selection of Yeast Cells with Gross-Chromosomal Rearrangements;503
31.3;Determining the Rate of Accumulating Gross-Chromosomal Rearrangements;504
31.4;Mapping and Sequencing of Chromosome V Breakpoints;506
31.5;Chromosome V Rearrangement Types;510
31.6;Conclusion;512
31.7;References;514
32;Chapter 28: Formation and Processing of Stalled Replication Forks-Utility of Two-Dimensional Agarose Gels;515
32.1;Introduction;515
32.2;Protocol;517
32.3;Interpretation;519
32.4;Closing Comments;527
32.5;Acknowledgments;529
32.6;References;529
33;Chapter 29: Poly(ADP-ribose) Polymerase-1 Activation During DNA Damage and Repair;531
33.1;Affinity Purification of Recombinant DNA-Damage Dependent Poly(ADP-Ribose) Polymerases (PARPs);532
33.2;PARP-1 Activation and DNA Damage;535
33.3;PARP-1 and DNA Repair;538
33.4;Acknowledgments;547
33.5;References;547
34;Chapter 30: Tyrosyl-DNA Phosphodiesterase (Tdp1) (3'-Phosphotyrosyl DNA Phosphodiesterase);549
34.1;Introduction;549
34.2;Tdp1;550
34.3;DNA Substrates;551
34.4;Kinetic Analysis;556
34.5;DNA Binding;559
34.6;References;561
35;Chapter 31: Assaying Double-Strand Break Repair Pathway Choice in Mammalian Cells Using a Targeted Endonuclease or the RAG Recombinase;562
35.1;Introduction;563
35.2;Materials;568
35.3;Methods;570
35.4;Notes;577
35.5;Acknowledgments;577
35.6;References;577
36;Author Index;579
37;Subject Index;601