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About this book
About this book
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. It will serve as the 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.
Part I. Mutant Isolation and Gene Cloning. Isolation of DNA Structure-dependent Checkpoint Mutants in S. pombe Rui G. Martinho and Antony M. Carr. Isolating Mutants of the Nematode Caenorhabditis elegans that are Hypersensitive to DNA-damaging Agents Phil S. Hartman and Naoaki Ishii. Isolating DNA Repair Mutants of Drosophila melanogaster Daryl S. Henderson. Generation Identification and Characterization of Repair-defective Mutants of Arabidopsis Anne Britt and Cai-Zhong Jiang. Screening for g-ray Hypersensitive Mutants of Arabidopsis Corinne Davies. Isolation of Mutagen- sensitive Chinese Hamster Cell Lines by Replica Plating Margaret Zdzienicka. Strategies for Cloning Mammalian DNA Repair Genes Larry H. Thompson. Novel Complementation Assays for DNA Repair Deficient Cells: Transient and Stable Expression of DNA Repair Genes Lin Zeng Alain Sarasin and Mauro Mezzina. Part II. Recognition and Removal of Inappropriate or Damaged DNA Bases. The Use of Electrophoretic Mobility Shift Assays to Study DNA Repair Byung Joon Hwang Vaughn Smider and Gilbert Chu. Mismatch Repair Assay Stephanie Corrette-Bennett/ Robert S. Lahue. Measurement of Activities of Cyclobutane-pyrimidine-dimer and (6-4)-photoproduct Photolyases. A Dot Blot Immunoassay for UV Photoproducts Shirley McCready. Measurement of Ultraviolet Radiation-induced DNA Damage Using Specific Antibodies Ann Stapleton. Quantification of DNA Photoproducts in Mammalian Cell DNA Using Radioimmunoassay David Mitchell. Monitoring Removal of Cyclobutane Pyrimidine Dimers in Arabidopsis John B. Hays/ Qishen Pang. DNA Damage Quantitation by Alkaline Gel Electrophoresis Betsy M. Sutherland Paula V. Bennett and John C. Sutherland. The Single Cell Gel Test (Comet Assay): A Sensitive Genotoxicity Test for the Detection of DNA Damage and Repair G?nter Speit and Andreas Hartmann. Measuring the Formation and Repair of UV Photoproducts by Ligation-mediated PCR Gerd P. Pfeifer and Reinhard Dammann. PCR-based Repair Assays for Strand-specific Measurement of DNA Damage and Repair I. Strand-specific quantitative PCR Keith A. Grimaldi/ John P. Bingham/ John A. Hartley. PCR-based Repair Assays for Strand-specific Measurement of DNA Damage and Repair II. Single Strand Ligation-PCR Keith A. Grimaldi Simon R. McAdam and John A. Hartley. Gene-specific and Mitochondrial Repair of Oxidative DNA Damage Michael R. Ansan and Vilhelm A. Bohr. Characterization of DNA Strand Cleavage by Enzymes That Act at Abasic Sites in DNA Walter Deutsch and Adly Yacoub. Base Excision repair Assay Using Xenopus laevis Oocyte Extracts Yoshihiro Matsumoto. In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts Guido Frosina Enrico Cappelli Paola Fortini Eugenia Dogliotti. Nucleotide Excision Repair in Saccharomyces cerevisiae Whole Cell Extracts Johnson M.S. Wong Zhigang He and C. James Ingles. In Vitro Excision Repair Assay in Schizosaccharomyces pombe Bernard Salles and Patrick Calsou. Nucleotide Excision Repair Assay in Drosophila melanogaster Using Established Cell Lines Kenji Kohno and Takuya Shimamoto. Nucleotide Excision Repair in Nuclear Extracts from Xenopus oocytes Eric J. Ackerman Lilian K. Koriazova Jitendra K. Saxena and Alexander Y. Spoonde. Assay for Nucleotide Excision Repair Protein Activity Using Fractionated Cell Extracts and UV-damaged Plasmid DNA Maureen Biggerstaff and Richard D. Wood. Dual Incision Assays for 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. In Vitro Chemiluminescence Assay to Measure Excision Repair in Cell Extracts. Bernard Salles and Christian Provot. Part III. DNA Strand Breakage and Repair. Physical Monitoring of HO-induced Homologous Recombination. Allyson Holmes and James E. Haber. Use of P-element Transposons to Study DNA Double-strand Break Repair in Drosophila melanogaster Daryl S. Henderson. Analyzing Double-strand Repair Events in Drosophila melanogaster. Gregory Gloor Tammy Dray and Kathy Keeler. Expression of I-Sce I in Drosophila to Induce DNA Double-strand Breaks Vladic Mogila Yohanns Bellaiche and Norbert Perrimon. Use of I-Sce I to Induce DNA Double-strand Breaks in Nicotiana Holger Puchta. Chromosomal Double Strand Breaks Introduced into Mammalian Cells by Expression of I-Sce I Endonuclease. Christine Richardson Beth Elliot and Maria Jasin. Induction of DNA Double-strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells James P. Carney and William F. Morgan. In Vitro Rejoining of Double-strand Breaks in Genomic DNA George Iliakis and Nge Cheong. Extrachromosomal Assay for DNA Double-strand Break Repair Feng Liang and Maria Jasin. Use of Gene Targeting to Study Recombination in Mammalian DNA Repair Mutants Rodney S. Nairn and Gerald M. Adair. Measurement of Low Frequency DNA Breaks Using Nucleoid Flow Cytometry Andrew Vaughan Scott Walter and Anne Milner. Part IV. DNA Damage Tolerance Mechanisms and Regulatory Responses. Live Analysis of the Division Cycles in X-irradiated Drosophila Embryos John Cunniff Justin Blethrow and William Sullivan. Inhibition of DNA Synthesis by Ionizing Radiation Margaret Zdzienicka and Nicolaas Jaspers. 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. Assays of Bypass Replication of Genotoxic Lesions in Mammalian Disease Mutant Cell- free Extracts Dan Svoboda and Jean-Michel H. Vos. Detection of DNA-bound PCNA in Cultured Cells Following Exposure to DNA Damaging Agents Masahiko Miura and Takehito Sasaki. Induction of p53 Protein as a Marker for Ionizing Radiation Exposure In Vivo David E. MacCallum and Ted R. Hupp. Activation of p53 Protein Function in Response to Cellular Irradiation. Jeremy P. Blaydes Alison Sparks and Ted R. Hupp. Selective Extraction of Fragmented DNA from Apoptotic Cells for Analysis by Gel Electrophoresis and Identification of Apoptotic Cells by Flow Cytometry Zbigniew Darzynkiewicz and Gloria Juan. Detection of DNA Strand Breakage in the Analysis of Apoptosis and Cell Proliferation by Flow and Laser Scanning Cytometry Zbigniew Darzynkiewicz. Immunoassay for Single-stranded DNA in Apoptotic Cells Oskar Frankfurt. Index.