Methods in Enzymology Volume 408 DNA Repair Part A 1st Edition by Judith Campbell, Paul Modrich 0121828131 9780121828134

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ISBN 10: 0121828131

ISBN 13: 9780121828134

Author: Judith L. Campbell, Paul Modrich

This volume 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.

Methods in Enzymology Volume 408 DNA Repair Part A 1st Table of contents:

Chapter 1: Assays for Determining Lesion Bypass Efficiency and Mutagenicity of Site-Specific DNA Les
Introduction
Genome Construction
Genome Normalization
Lesion Bypass (CRAB) Assay
Lesion Mutagenesis (REAP) Assay
Conclusion
References

Chapter 2: Oxidative DNA Glycosylases: Recipes from Cloning to Characterization
Introduction
Purification of DNA Glycosylases
Preparation of Oligonucleotide Substrates
Activity Assays for Oxidative DNA Glycosylases
Acknowledgment
References

Chapter 3: Purification and Characterization of NEIL1 and NEIL2, Members of a Distinct Family of Mam
Endogenous and Induced DNA Damage
Repair of Oxidized Base Lesions in the Genome
Two Classes of Oxidized Base-Specific Glycosylases
Human Genome Database Analysis and Discovery of MutM/Nei-Like Proteins
Enzymatic Activity of NEIL1 and NEIL2 with Duplex or Bubble Oligonucleotide Substrates
Detection of DNA Glycosylase Activity of Recombinant NEILs by Trapping Analysis
Acknowledgments
References

Chapter 4: Analysis of Base Excision DNA Repair of the Oxidative Lesion 2-Deoxyribonolactone and the
Introduction
Preparation of dL-DNA Substrates
Analysis of dL-mediated Spontaneous DNA-Protein Cross-Links
Analysis of dL-Specific BER
Acknowledgments
References

Chapter 5: Isolation and Analyses of MutY Homologs (MYH)
Introduction
Isolation of MutY and MYH Proteins
Functional Assays of MutY and MYH
Protein Interaction Assays
Acknowledgments
References

Chapter 6: Use of Yeast for Detection of Endogenous Abasic Lesions, Their Source, and Their Repair
AP Sites in DNA and Related 3′-Blocked and 5′-Blocked Single Strand Breaks Form an Abundant and Dele
Endogenous AP Sites Cause Cell Death in the Absence of Apn1, Apn2, and Rad1-Rad10
Use of the apn1 apn2 rad1 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA
Use of the apn1 apn2 rad14 Triple Mutant to Study the Origin and Repair of Endogenous AP Sites in DNA
Conclusions and Model
Acknowledgments
References

Chapter 7: Activities and Mechanism of DNA Polymerase Beta
Introduction
Kinetic Characteristics of DNA Synthesis
Measurement of dRP Lyase Activity
Measurement of In Vitro BER
Reagents
Acknowledgment
References

Chapter 8: Direct Removal of Alkylation Damage from DNA by AlkB and Related DNA Dioxygenases
Purification of AlkB, ABH2, and ABH3
14C-Methylated Substrates
Assays
In Vivo Assay: Survival of Alkylated Single-Stranded DNA Bacteriophage in E. coli AlkB Mutants
References

Chapter 9: Purification and Characterization of DNA Photolyases
Introduction
Overexpression and Purification of Photolyases
Spectroscopic Properties and Characterization of Purified Photolyases
Assays for DNA Binding and Repair by Photolyase
Acknowledgments
References

Chapter 10: Genetic and In Vitro Assays of DNA Deamination
Introduction
Escherichia coli Assay of Deamination
In Vitro Assay of Deamination
Conclusion
Acknowledgments
References

Chapter 11: The Xeroderma Pigmentosum Group C Protein Complex and Ultraviolet-Damaged DNA-Binding Proteins
Introduction
Purification of Recombinant Proteins
Damage-Specific DNA-Binding Assays
In Vitro NER Assays
UV-Induced Ubiquitylation Mediated by the UV-DDB-E3 Complex
References

Chapter 12: Purification and Characterization of Escherichia coli and Human Nucleotide Excision Repair Proteins
Introduction
Substrates for In Vitro Repair Assays
Enzymes for Repair Assays
In Vitro Repair Assays
Concluding Comments
Acknowledgment
References

Chapter 13: Assays for Transcription Elongation by RNA Polymerase II Using Oligo(dC)-Tailed Templates
Introduction
Construction of Closed-Circular Duplex DNA Substrates Containing a DNA Lesion at a Specific Site
Preparation of an Oligo(dC)-Tailed Template from Closed-Circular Duplex DNA Substrates
RNAPII Elongation Reaction Using Oligo(dC)-Tailed Template
Hot Labeling Reaction
Cold Labeling Reaction
Preparation of RNA Transcripts from RNAPII Transcription Elongation Assay Using Oligo(dC)-Tailed Template
Analysis of Lesion-Bypassed RNAPII Elongation Transcripts
Reverse-Transcription Reaction for First-Strand cDNA Synthesis and PCR
Analysis of 3′ Ligation-Mediated RT-PCR
Acknowledgments
References

Chapter 14: In Vivo Assays for Transcription-Coupled Repair
Introduction
Important General Considerations
Southern Blot Method
Ligation-Mediated PCR Method
Acknowledgments
References

Chapter 15: TFIIH Enzymatic Activities in Transcription and Nucleotide Excision Repair
Introduction
Purification of Human TFIIH from Wild-Type and Patient Cell Lines
DNA Substrate Preparation Procedure
Enzymatic Assays
Acknowledgments
References

Chapter 16: An Assay for Studying Ubiquitylation of RNA Polymerase II and Other Proteins in Crude Yeast Extracts
Introduction
Whole Cell Extract Preparation
Preparation of myc-Tagged Ubiquitin
Purification of RNA Polymerase II
In Vitro Ubiquitylation
References

Chapter 17: Analysis of the Excision Step in Human DNA Mismatch Repair
Introduction
Methods
Discussion
Acknowledgments
References

Chapter 18: Characterization of the “Mismatch Repairosome” and Its Role in the Processing of Modified DNA
Introduction
Construction of Substrates Containing Insertion/Deletion Loops
Construction of G/T and G/C Mismatch Repair Substrates
Preparation of Nuclear Extracts
In Vitro Mismatch Repair Assays
DNA Affinity Purification
Conclusions
Acknowledgments

Chapter 19: Analysis of DNA Mismatch Repair in Cellular Response to DNA Damage
Introduction
In Vitro Biochemistry Studies
Studies at Cellular Level
Animal Models
Acknowledgments
References

Chapter 20: Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors
Introduction
Purification and Characterization of umuD Gene Products
Characterization of Escherichia coli Y Family Polymerases In Vitro and In Vivo
Conclusions
Acknowledgments
References

Chapter 21: Measuring the Fidelity of Translesion DNA Synthesis
Introduction
Experimental Outline of Translesion Synthesis Fidelity Assays
Materials
Experimental Procedures
Applications of the Method
Acknowledgments
References

Chapter 22: DNA Polymerases for Translesion DNA Synthesis: Enzyme Purification and Mouse Models for Genetic Studies
Introduction
Purification of Specialized DNA Polymerases as Tagged Proteins Following Expression of Cloned Fusion Genes
Mouse Strains Defective in Specialized DNA Polymerases
References

Chapter 23: Purification and Characterization of Escherichia coli DNA Polymerase V
Introduction
Assay Methods
Purification of DNA Polymerase V
Acknowledgments
References

Chapter 24: Yeast and Human Translesion DNA Synthesis Polymerases: Expression, Purification, and Biochemical Characterization
Introduction
Expression of GST-Tagged TLS Polymerases in Yeast
Protein Purification
DNA Synthesis Assays
Special Considerations for Individual TLS Polymerases
Acknowledgment
References

Chapter 25: Localization of Y-Family Polymerases and the DNA Polymerase Switch in Mammalian Cells
Introduction
Cellular Localization of Translesion Synthesis Polymerases
Ubiquitination of PCNA and Interaction with POLη
References

Chapter 26: Repair of DNA Double Strand Breaks: In Vivo Biochemistry
Introduction
Methods
Media
Galactose Induction
DNA Extraction
Southern Analysis Using Native and Denaturing Gels
Analysis of Single-Stranded Tail Formation Using Slot Blots
Polymerase Chain Reaction Analysis
Chromatin Immunoprecipitation Procedure
Comments
References

Chapter 27: Assays for Nonhomologous End Joining in Extracts
Introduction
Preparation of Human Whole Cell Extracts That Support NHEJ
Gel-Based Assay for NHEJ of Compatible DNA Ends
Quantitative PCR Assay for NHEJ of Compatible or Noncompatible DNA Ends
Analysis of Processing Events
Concluding Remarks
Acknowledgments
References

Chapter 28: Purification and Assays of Saccharomyces cerevisiae Homologous Recombination Proteins
Introduction
Expression and Purification of Homologous Recombination Proteins
Biochemical Systems for Studying Homologous DNA Pairing and Strand Exchange
Acknowledgment
References

Chapter 29: Analysis of DNA Recombination and Repair Proteins in Living Cells by Photobleaching Microscopy
Introduction
Fluorescence Recovery after Photobleaching
Experimental Approach
Conclusions
Acknowledgments
References

Chapter 30: Synthetic Junctions as Tools to Identify and Characterize Holliday Junction Resolvases
Introduction
Preparation of Synthetic Holliday Junctions
Holliday Junction Resolution Assays
Acknowledgments
References

Chapter 31: In Vitro Nonhomologous DNA End Joining System
Introduction
Procedures
General Remarks
Concluding Remarks
References

Chapter 32: RAG and HMGB1 Proteins: Purification and Biochemical Analysis of Recombination Signal Complexes
Introduction
Expression Vectors
Protein Expression and Purification
Oligonucleotide Probe Preparation
In Vitro Cleavage Assays
Electrophoretic Mobility Shift Assays (EMSA)
In-Gel Cleavage Assays
Discussion
Acknowledgments
References

Chapter 33: Purification and Biochemical Characterization of Ataxia-Telangiectasia Mutated and Mre11-Rad50-Nbs1 Complex
Introduction
Purification of Dimeric ATM
Purification of Monomeric ATM
Anti-Flag Affinity Chromatography
Purification of the MRN Complex
ATM Kinase Assay
References

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