In DNA Repair Protocols: Eukaryotic Systems, Daryl S. Henderson and a team of hands-on experts give time-tested instructions for analyzing a wide range of DNA repair processes and cellular responses to DNA damage, including nucleotide and base excision repair, DNA strand break repair, and mismatch repair. The methods focus on eukaryotic model systems that have made, or have the potential to make, important contributions to our understanding of cellular responses to DNA damage in relation to mutagenesis, carcinogenesis, and the cell cycle. Although mammalian cells predominate, consideration is…mehr
In DNA Repair Protocols: Eukaryotic Systems, Daryl S. Henderson and a team of hands-on experts give time-tested instructions for analyzing a wide range of DNA repair processes and cellular responses to DNA damage, including nucleotide and base excision repair, DNA strand break repair, and mismatch repair. The methods focus on eukaryotic model systems that have made, or have the potential to make, important contributions to our understanding of cellular responses to DNA damage in relation to mutagenesis, carcinogenesis, and the cell cycle. Although mammalian cells predominate, consideration is also given to such important nonmammalian model systems as yeast, C. elegans (nematode), Drosophila (fruitfly), Xenopus (amphibian), and plants.
DNA Repair Protocols: Eukaryotic Systems offers the most comprehensive collection of DNA repair protocols available, all step-by-step, optimized, and eminently reproducible with tips on troubleshooting and avoiding pitfalls. It will serve asthe gold-standard reference for both the practical and theoretical aspects of DNA repair studies, encourage the transfer of methodologies between model systems, and stimulate the development of new approaches.
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I. Mutant Isolation and Gene Cloning.- 1 Isolation of DNA Structure-Dependent Checkpoint Mutants in S. pombe.- 2 Isolating Mutants of the Nematode Caenorhabditis elegans That Are Hypersensitive to DNA-Damaging Agents.- 3 Isolating DNA Repair Mutants of Drosophila melanogaster.- 4 Generation, Identification, and Characterization of Repair-Defective Mutants of Arabidopsis.- 5 Screening for ?-Ray Hypersensitive Mutants of Arabidopsis.- 6 Isolation of Mutagen-Sensitive Chinese Hamster Cell Lines by Replica Plating.- 7 Strategies for Cloning Mammalian DNA Repair Genes.- 8 Novel Complementation Assays for DNA Repair-Deficient Cells: Transient and Stable Expression of DNA Repair Genes.- II. Recognition and Removal of Inappropriate or Damaged DNA Bases.- 9 The Use of Electrophoretic Mobility Shift Assays to Study DNA Repair.- 10 Mismatch Repair Assay.- 11 Measurement of Activities of Cyclobutane-Pyrimidine-Dimer and (6-4)-Photoproduct Photolyases.- 12 A Dot Blot Immunoassay for UV Photoproducts.- 13 Measurement of UV Radiation-Induced DNA Damage Using Specific Antibodies.- 14 Quantification of Photoproducts in Mammalian Cell DNA Using Radioimmunoassay.- 15 Monitoring Removal of Cyclobutane Pyrimidine Dimers in Arabidopsis.- 16 DNA Damage Quantitation by Alkaline Gel Electrophoresis.- 17 The Comet Assay (Single-Cell Gel Test): A Sensitive Genotoxicity Test for the Detection of DNA Damage and Repair.- 18 Measuring the Formation and Repair of UV Photoproducts by Ligation-Mediated PCR.- 19 PCR-Based Assays for Strand-Specific Measurement of DNA Damage and Repair I: Strand-Specific Quantitative PCR.- 20 PCR-Based Assays for Strand-Specific Measurement of DNA Damage and Repair II: Single-Strand Ligation-PCR.- 21 Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage.- 22Characterization of DNA Strand Cleavage by Enzymes That Act at Abasic Sites in DNA.- 23 Base Excision Repair Assay Using Xenopus laevis Oocyte Extracts.- 24 In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts.- 25 Nucleotide Excision Repair in Saccharomyces cerevisiae Whole-Cell Extracts.- 26 In Vitro Excision Repair Assay in Schizosaccharomyces pombe.- 27 Nucleotide Excision Repair Assay in Drosophila melanogaster Using Established Cell Lines.- 28 Nucleotide Excision Repair in Nuclear Extracts from Xenopus Oocytes.- 29 Assay for Nucleotide Excision Repair Protein Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA.- 30 Dual-Incision Assays for Nucleotide Excision Repair Using DNA with a Lesion at a Specific Site.- 31 In Vitro Chemiluminescence Assay to Measure Excision Repair in Cell Extracts.- III. DNA Strand Breakage and Repair.- 32 Physical Monitoring of HO-Induced Homologous Recombination.- 33 Use of P Element Transposons to Study DNA Double-Strand Break Repair in Drosophila melanogaster.- 34 Analyzing Double-Strand Repair Events in Drosophila.- 35 Expression of I-Sce I in Drosophila to Induce DNA Double-Strand Breaks.- 36 Use of I-Sce I to Induce DNA Double-Strand Breaks in Nicotiana.- 37 Chromosomal Double-Strand Breaks Introduced in Mammalian Cells by Expression of I-Sce I Endonuclease.- 38 Induction of DNA Double-Strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells.- 39 In Vitro Rejoining of Double-Strand Breaks in Genomic DNA.- 40 Extrachromosomal Assay for DNA Double-Strand Break Repair.- 41 Use of Gene Targeting to Study Recombination in Mammalian DNA Repair Mutants.- 42 Measurement of Low-Frequency DNA Breaks Using Nucleoid Flow Cytometry.- IV. DNA Damage Tolerance Mechanisms and Regulatory Responses.- 43 Live Analysis of the Division Cycles in X-Irradiated Drosophila Embryos.- 44 Inhibition of DNA Synthesis by Ionizing Radiation.- 45 Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40-Based In Vitro Assay of DNA Replication.- 46 Assays of Bypass Replication of Genotoxic Lesions in Mammalian Disease and Mutant Cell-Free Extracts.- 47 Detection of Chromatin-Bound PCNA in Cultured Cells Following Exposure to DNA-Damaging Agents.- 48 Induction of p53 Protein as a Marker for Ionizing Radiation Exposure In Vivo.- 49 Activation of p53 Protein Function in Response to Cellular Irradiation.- 50 Selective Extraction of Fragmented DNA from Apoptotic Cells for Analysis by Gel Electrophoresis and Identification of Apoptotic Cells by Flow Cytometry.- 51 Detection of DNA Strand Breakage in the Analysis of Apoptosis and Cell Proliferation by Flow and Laser Scanning Cytometry.- 52 Immunoassay for Single-Stranded DNA in Apoptotic Cells.
Isolation of Mutagen-Sensitive Chinese Hamster Cell Lines by Replica Plating Malgorzata Z. Zdzienicka Complementation Assays Adapted for DNA Repair-Deficient Keratinocytes Mathilde Fréchet, Valérie Bergoglio, Odile Chevallier-Lagente, Alain Sarasin, and Thierry Magnaldo Cytogenetic Challenge Assays for Assessment of DNA Repair Capacities William W. Au and Salama A. Salama Evaluating the Delayed Effects of Cellular Exposure to Ionizing Radiation Shruti Nagar, James J. Corcoran, and William F. Morgan Inhibition of DNA Synthesis by Ionizing Radiation: A Marker for an S-Phase Checkpoint Nicolaas G. J. Jaspers and Malgorzata Z. Zdzienicka Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40-Based In Vitro Assay of DNA Replication George Iliakis, Ya Wang, and Hong Yan Wang Cytometric Assessment of Histone H2AX Phosphorylation: A Reporter of DNA Damage Xuan Huang and Zbigniew Darzynkiewicz Detection of DNA Strand Breaks by Flow and Laser Scanning Cytometry in Studies of Apoptosis and Cell Proliferation (DNA Replication) Zbigniew Darzynkiewicz, Xuan Huang, and Masaki Okafugi In Vitro Rejoining of Double-Strand Breaks in Genomic DNA George Iliakis and Nge Cheong Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR Michael J. Villalobos, Christopher J. Betti, and Andrew T. M Vaughan Plasmid-Based Assays for DNA End-Joining In Vitro George Iliakis, Bustanur Rosidi, Minli Wang, and Huichen Wang Use of Gene Targeting to Study Recombination in Mammalian Cell DNA Repair Mutants Rodney S. Nairn and Gerald M. Adair Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage R. Michael Anson, Penelope A. Mason, and Vilhelm A. Bohr Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells Janine H. Santos, Joel N. Meyer, Bhaskar S. Mandavilli, and Bennett Van Houten Measuring the Formation and Repair of DNA Damage by Ligation-Mediated PCR GerdP. Pfeifer Immunochemical Detection of UV-Induced DNA Damage and Repair Marcus S. Cooke and Alistair Robson A Dot-Blot Immunoassay for Measuring Repair of Ultraviolet Photoproducts Shirley McCready Quantification of Photoproducts in Mammalian Cell DNA Using Radioimmunoassay David L. Mitchell DNA Damage Quantitation by Alkaline Gel Electrophoresis Betsy M. Sutherland, Paula V. Bennett, and John C. Sutherland The Comet Assay: A Sensitive Genotoxicity Test for the Detection of DNA Damage and Repair Günter Speit and Andreas Hartmann Fast Micromethod DNA Single-Strand-Break Assay Heinz C. Schröder, Renato Batel, Heiko Schwertner, Oleksandra Boreiko, and Werner E. G. Müller 32P-Postlabeling DNA Damage Assays: PAGE, TLC, and HPLC Shinya Shibutani, Sung Yeon Kim, and Naomi Suzuki Electrophoretic Mobility Shift Assays to Study Protein Binding to Damaged DNA Vaughn Smider, Byung Joon Hwang, and Gilbert Chu Construction of MMR Plasmid Substrates and Analysis of MMR Error Correction and Excision Huixian Wang and John B. Hays Characterization of Enzymes that Initiate Base Excision Repair at Abasic Sites Walter A. Deutsch and Vijay Hegde Base Excision Repair in Mammalian Cells Yoshihiro Matsumoto In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts Guido Frosina, Enrico Cappelli, Monica Ropolo, Paola Fortini, Barbara Pascucci, and Eugenia Dogliotti Biochemical Assays for the Characterization of DNA Helicases Robert M. Brosh, Jr. and Sudha Sharma Repair Synthesis Assay for Nucleotide Excision Repair Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA Maureen Biggerstaff and Richard D. Wood Assaying for the Dual Incisions of Nucleotide Excision Repair Using DNA with a Lesion at a Specific Site Mahmud K. K. Shivji, Jonathan G. Moggs, Isao Kuraoka, and Richard D. Wood Analysis of Proliferating Cell Nuclear Antigen (PCNA) Associated with DNA Excision Repair Sites in Mammalian Cells A. Ivana Scovassi
I. Mutant Isolation and Gene Cloning.- 1 Isolation of DNA Structure-Dependent Checkpoint Mutants in S. pombe.- 2 Isolating Mutants of the Nematode Caenorhabditis elegans That Are Hypersensitive to DNA-Damaging Agents.- 3 Isolating DNA Repair Mutants of Drosophila melanogaster.- 4 Generation, Identification, and Characterization of Repair-Defective Mutants of Arabidopsis.- 5 Screening for ?-Ray Hypersensitive Mutants of Arabidopsis.- 6 Isolation of Mutagen-Sensitive Chinese Hamster Cell Lines by Replica Plating.- 7 Strategies for Cloning Mammalian DNA Repair Genes.- 8 Novel Complementation Assays for DNA Repair-Deficient Cells: Transient and Stable Expression of DNA Repair Genes.- II. Recognition and Removal of Inappropriate or Damaged DNA Bases.- 9 The Use of Electrophoretic Mobility Shift Assays to Study DNA Repair.- 10 Mismatch Repair Assay.- 11 Measurement of Activities of Cyclobutane-Pyrimidine-Dimer and (6-4)-Photoproduct Photolyases.- 12 A Dot Blot Immunoassay for UV Photoproducts.- 13 Measurement of UV Radiation-Induced DNA Damage Using Specific Antibodies.- 14 Quantification of Photoproducts in Mammalian Cell DNA Using Radioimmunoassay.- 15 Monitoring Removal of Cyclobutane Pyrimidine Dimers in Arabidopsis.- 16 DNA Damage Quantitation by Alkaline Gel Electrophoresis.- 17 The Comet Assay (Single-Cell Gel Test): A Sensitive Genotoxicity Test for the Detection of DNA Damage and Repair.- 18 Measuring the Formation and Repair of UV Photoproducts by Ligation-Mediated PCR.- 19 PCR-Based Assays for Strand-Specific Measurement of DNA Damage and Repair I: Strand-Specific Quantitative PCR.- 20 PCR-Based Assays for Strand-Specific Measurement of DNA Damage and Repair II: Single-Strand Ligation-PCR.- 21 Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage.- 22Characterization of DNA Strand Cleavage by Enzymes That Act at Abasic Sites in DNA.- 23 Base Excision Repair Assay Using Xenopus laevis Oocyte Extracts.- 24 In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts.- 25 Nucleotide Excision Repair in Saccharomyces cerevisiae Whole-Cell Extracts.- 26 In Vitro Excision Repair Assay in Schizosaccharomyces pombe.- 27 Nucleotide Excision Repair Assay in Drosophila melanogaster Using Established Cell Lines.- 28 Nucleotide Excision Repair in Nuclear Extracts from Xenopus Oocytes.- 29 Assay for Nucleotide Excision Repair Protein Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA.- 30 Dual-Incision Assays for Nucleotide Excision Repair Using DNA with a Lesion at a Specific Site.- 31 In Vitro Chemiluminescence Assay to Measure Excision Repair in Cell Extracts.- III. DNA Strand Breakage and Repair.- 32 Physical Monitoring of HO-Induced Homologous Recombination.- 33 Use of P Element Transposons to Study DNA Double-Strand Break Repair in Drosophila melanogaster.- 34 Analyzing Double-Strand Repair Events in Drosophila.- 35 Expression of I-Sce I in Drosophila to Induce DNA Double-Strand Breaks.- 36 Use of I-Sce I to Induce DNA Double-Strand Breaks in Nicotiana.- 37 Chromosomal Double-Strand Breaks Introduced in Mammalian Cells by Expression of I-Sce I Endonuclease.- 38 Induction of DNA Double-Strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells.- 39 In Vitro Rejoining of Double-Strand Breaks in Genomic DNA.- 40 Extrachromosomal Assay for DNA Double-Strand Break Repair.- 41 Use of Gene Targeting to Study Recombination in Mammalian DNA Repair Mutants.- 42 Measurement of Low-Frequency DNA Breaks Using Nucleoid Flow Cytometry.- IV. DNA Damage Tolerance Mechanisms and Regulatory Responses.- 43 Live Analysis of the Division Cycles in X-Irradiated Drosophila Embryos.- 44 Inhibition of DNA Synthesis by Ionizing Radiation.- 45 Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40-Based In Vitro Assay of DNA Replication.- 46 Assays of Bypass Replication of Genotoxic Lesions in Mammalian Disease and Mutant Cell-Free Extracts.- 47 Detection of Chromatin-Bound PCNA in Cultured Cells Following Exposure to DNA-Damaging Agents.- 48 Induction of p53 Protein as a Marker for Ionizing Radiation Exposure In Vivo.- 49 Activation of p53 Protein Function in Response to Cellular Irradiation.- 50 Selective Extraction of Fragmented DNA from Apoptotic Cells for Analysis by Gel Electrophoresis and Identification of Apoptotic Cells by Flow Cytometry.- 51 Detection of DNA Strand Breakage in the Analysis of Apoptosis and Cell Proliferation by Flow and Laser Scanning Cytometry.- 52 Immunoassay for Single-Stranded DNA in Apoptotic Cells.
Isolation of Mutagen-Sensitive Chinese Hamster Cell Lines by Replica Plating Malgorzata Z. Zdzienicka Complementation Assays Adapted for DNA Repair-Deficient Keratinocytes Mathilde Fréchet, Valérie Bergoglio, Odile Chevallier-Lagente, Alain Sarasin, and Thierry Magnaldo Cytogenetic Challenge Assays for Assessment of DNA Repair Capacities William W. Au and Salama A. Salama Evaluating the Delayed Effects of Cellular Exposure to Ionizing Radiation Shruti Nagar, James J. Corcoran, and William F. Morgan Inhibition of DNA Synthesis by Ionizing Radiation: A Marker for an S-Phase Checkpoint Nicolaas G. J. Jaspers and Malgorzata Z. Zdzienicka Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40-Based In Vitro Assay of DNA Replication George Iliakis, Ya Wang, and Hong Yan Wang Cytometric Assessment of Histone H2AX Phosphorylation: A Reporter of DNA Damage Xuan Huang and Zbigniew Darzynkiewicz Detection of DNA Strand Breaks by Flow and Laser Scanning Cytometry in Studies of Apoptosis and Cell Proliferation (DNA Replication) Zbigniew Darzynkiewicz, Xuan Huang, and Masaki Okafugi In Vitro Rejoining of Double-Strand Breaks in Genomic DNA George Iliakis and Nge Cheong Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR Michael J. Villalobos, Christopher J. Betti, and Andrew T. M Vaughan Plasmid-Based Assays for DNA End-Joining In Vitro George Iliakis, Bustanur Rosidi, Minli Wang, and Huichen Wang Use of Gene Targeting to Study Recombination in Mammalian Cell DNA Repair Mutants Rodney S. Nairn and Gerald M. Adair Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage R. Michael Anson, Penelope A. Mason, and Vilhelm A. Bohr Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells Janine H. Santos, Joel N. Meyer, Bhaskar S. Mandavilli, and Bennett Van Houten Measuring the Formation and Repair of DNA Damage by Ligation-Mediated PCR GerdP. Pfeifer Immunochemical Detection of UV-Induced DNA Damage and Repair Marcus S. Cooke and Alistair Robson A Dot-Blot Immunoassay for Measuring Repair of Ultraviolet Photoproducts Shirley McCready Quantification of Photoproducts in Mammalian Cell DNA Using Radioimmunoassay David L. Mitchell DNA Damage Quantitation by Alkaline Gel Electrophoresis Betsy M. Sutherland, Paula V. Bennett, and John C. Sutherland The Comet Assay: A Sensitive Genotoxicity Test for the Detection of DNA Damage and Repair Günter Speit and Andreas Hartmann Fast Micromethod DNA Single-Strand-Break Assay Heinz C. Schröder, Renato Batel, Heiko Schwertner, Oleksandra Boreiko, and Werner E. G. Müller 32P-Postlabeling DNA Damage Assays: PAGE, TLC, and HPLC Shinya Shibutani, Sung Yeon Kim, and Naomi Suzuki Electrophoretic Mobility Shift Assays to Study Protein Binding to Damaged DNA Vaughn Smider, Byung Joon Hwang, and Gilbert Chu Construction of MMR Plasmid Substrates and Analysis of MMR Error Correction and Excision Huixian Wang and John B. Hays Characterization of Enzymes that Initiate Base Excision Repair at Abasic Sites Walter A. Deutsch and Vijay Hegde Base Excision Repair in Mammalian Cells Yoshihiro Matsumoto In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts Guido Frosina, Enrico Cappelli, Monica Ropolo, Paola Fortini, Barbara Pascucci, and Eugenia Dogliotti Biochemical Assays for the Characterization of DNA Helicases Robert M. Brosh, Jr. and Sudha Sharma Repair Synthesis Assay for Nucleotide Excision Repair Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA Maureen Biggerstaff and Richard D. Wood Assaying for the Dual Incisions of Nucleotide Excision Repair Using DNA with a Lesion at a Specific Site Mahmud K. K. Shivji, Jonathan G. Moggs, Isao Kuraoka, and Richard D. Wood Analysis of Proliferating Cell Nuclear Antigen (PCNA) Associated with DNA Excision Repair Sites in Mammalian Cells A. Ivana Scovassi
Rezensionen
"...It will serve as an essential reference for both the practical and theoretical aspects of DNA repair studies, encourage the transfer of methodologies between model systems, and stimulate the development of new approaches." - Anticancer Research"...must for any science library supporting a molecular biology program." - E-Streams"An informative collection of techniques and protocols for characterizing and utilizing DNA repair activities and enzymes from some of the leading names in DNA repair field. This collection of protocols is ideal for both the novice in the field and to the experienced applied researcher." - Lavoisier Librairie
"...an extremely useful resource for any laboratory with an interest in the vibrant field of DNA repair. Any good laboratory manueal should be not only a useful reference guide but also a source of inspiration, and it is undoubtedly true that DNA Repair Protocols: Mammalian Systems succeeds on both counts." -Int. J. Radiation Oncology Biol. Phys.
"This is the definite book on the subject not only for those working in the field of DNA repair but also for those interested in genetic toxicology and chemical safety." -BTS Newsletter
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