DNA Damage Responses: Methods and Protocols (Methods in Molecular Biology, 2444)

✍ Scribed by Nima Mosammaparast (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 285
• Category: Library

✦ Synopsis

This volume provides detailed methods and key approaches used to mechanistically study DNA damage, as well as the factors involved in the damage response. Chapters guide readers through proteomics and biophysical approaches, analyzing protein function, quantifying DNA replication dynamics and nucleic acid base damage, as well as biochemical reconstitution of key pathways involved in DNA repair. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, DNA Damage Responses: Methods and Protocols aims to be a useful practical guide to researches to help further their study in this field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Robust Computational Approaches to Defining Insights on the Interface of DNA Repair with Replication and Transcript...
1 Introduction
2 Materials
3 Methods
3.1 Compile C++ Programs
3.2 Search for Non-B DNA-Forming Sequences in Fasta Files
3.3 Assess TCGA Gene Expression Levels Between Tumor and Normal Controls
3.4 Kaplan-Meier Survival Curves in TCGA Patients with High and Low mRNA Levels
3.5 Gene Correlation Expression Analyses (GCEA)
3.6 Basic Utilities
4 Notes
References
Chapter 2: A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids
2.3 Antibodies
2.4 Other Reagents
2.5 PCR Primers
3 Methods
3.1 Generation of abl Pre-B Cell Lines
3.2 Establishing abl Pre-B Cell Line with Inducible Cas9 Using Lentiviral pCW-Cas9
3.3 Preparation of Lentiviral gRNA Library
3.4 Preparation of Genome-Scale abl Pre-B Cell gRNA Libraries
3.5 Genome-Scale Screen for Genes Regulating DNA DSB End Resection in G0/G1-Arrested Abl Pre-B Cells Using Flow Cytometry-Base...
3.6 PCR of gRNAs from Genomic DNA Isolated from Sorted Cells
4 Notes
References
Chapter 3: Immunoaffinity Purification of Epitope-Tagged DNA Repair Complexes from Human Cells
1 Introduction
2 Materials
2.1 Production of Stably Expressing Proteins in Mammalian Cell Lines
2.2 Cell Extract Preparation
2.3 Immunoaffinity Purification
3 Methods
3.1 Generation of Cell Lines Expressing Epitope-Tagged Protein of Interest
3.1.1 Transfection of Retroviral Vectors into Packaging Cells
3.1.2 Retroviral Transduction and Antibiotic Selection
3.1.3 Large-Scale Growth of Transduced Cells
3.2 Preparation of Nuclear Extracts
3.3 Immunoaffinity Purification
3.3.1 Anti-Flag Immunoaffinity Purification
3.3.2 Anti-HA Immunoaffinity Purification
3.3.3 Analysis of Affinity Purified Complexes
4 Notes
References
Chapter 4: Universally Accessible Structural Data on Macromolecular Conformation, Assembly, and Dynamics by Small Angle X-Ray ...
1 Introduction
2 Materials
3 Method
3.1 Obtaining SAXS Data Collection Time at the SIBYLS Beamline
3.2 HT-SAXS Sample Preparation
3.2.1 HT-SAXS Sample Preparation by Dialysis
3.2.2 HT-SAXS Sample Preparation by Concentrator
3.2.3 HT-SAXS Sample Preparation by SEC
3.3 SEC-SAXS Sample Preparation
3.4 HT-SAXS Data Averaging
3.5 SEC-SAXS Data Averaging
3.6 Calculation of Metrics from HT-SAXS or SEC-SAXS Data
3.7 Comparison of Atomic Models to Experimental SAXS Data by FOXS
3.8 Generation of Flexible Atomic Models to Compare Ensembles with Experimental SAXS Data by BILBOMD
3.9 Future Prospects
4 Notes
References
Chapter 5: Assessing DNA Damage Responses Using B Lymphocyte Cultures
1 Introduction
2 Materials
2.1 Cell Lines and Plasmids
2.2 Culture Media and Solutions
3 Methods
3.1 Generating Abelson Pre-B Cell Lines
3.1.1 Generation of V-abl-Expressing Retrovirus
3.1.2 Isolation of Hematopoietic Cells from Murine Bone Marrow
3.1.3 Transduction of Murine Hematopoietic Cells with V-abl Retrovirus
3.2 Establishing Primary Pre-B Cell Cultures
3.3 Induction of RAG-Mediated DSBs
3.3.1 RAG DSBs in Abl Pre-B Cells
3.3.2 RAG DSBs in Primary Pre-B Cells
3.4 Quantitation of DNA Breaks
3.4.1 Isolation of Genomic DNA
3.4.2 Quantitative PCR Protocol for Measuring DNA Breaks
3.5 Measurement of DNA Damage Responses
3.5.1 Protein and RNA Collection from Abl Pre-B Cells and Primary Pre-B Cells
3.5.2 Immunofluorescence of DNA Damage Foci in Abl Pre-B Cells and Primary Pre-B Cells
4 Notes
References
Chapter 6: Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy
1 Introduction
2 Materials
2.1 In-Cell Psoralen Cross-Linking and Lysis
2.2 Genomic DNA Extraction
2.3 DNA Digestion and Enrichment of Replication Intermediates
2.4 Carbon Coating of the Grids
2.5 Spreading of the DNA
2.6 Platinum Shadowing of Grids
2.7 Visualization of DNA Via Transmission Electron Microscope
2.8 Analysis of Single-Stranded DNA Gaps at the Replication Fork
3 Methods
3.1 In-Cell Psoralen Cross-Linking and Lysis
3.2 Genomic DNA Extraction
3.3 DNA Digestion and Enrichment of Replication Intermediates
3.4 Carbon Coating of the Grids
3.5 Spreading of DNA
3.6 Platinum Shadowing of Grids
3.7 Visualization of DNA Via Transmission Electron Microscope
3.8 Analysis of ssDNA Gaps at the Replication Fork
4 Notes
References
Chapter 7: Approaches to Monitor Termination of DNA Replication Using Xenopus Egg Extracts
1 Introduction
2 Materials
2.1 Immunodepletion of Xenopus Egg Extracts
2.2 Replication of Plasmid DNA in Xenopus Egg Extracts
2.3 Purification of DNA Replication Intermediates
2.4 Analysis of DNA Synthesis Using Native Agarose Gels
2.5 Analysis of Fork Merger Using Native Agarose Gels
2.6 Analysis of Ligation Using Denaturing Agarose Gels
2.7 Analysis of Replication Protein Binding by Chromatin Capture
3 Methods
3.1 Immunodepletion of Xenopus Egg Extracts
3.2 Replication of Plasmid DNA in Xenopus Egg Extracts
3.3 Purification of DNA Replication Intermediates
3.4 Separation of Replication Intermediates Using Native Agarose Gels
3.5 Analysis of DNA Synthesis and Decatenation Using Native Agarose Gels
3.6 Preparation of Replication Fork Structures
3.7 Separation of Replication Fork Structures Using a Native Agarose Gel
3.8 Analysis of Fork Merger Using Native Agarose Gels
3.9 Preparation of Nascent Strands
3.10 Separation of Nascent Strands Using a Denaturing Agarose Gel
3.11 Analysis of Ligation
3.12 Recovery of Chromatin-Associated Proteins
3.13 Detection of Chromatin Capture Samples by Western Blotting
3.14 Analysis of Replisome Unloading
4 Notes
References
Chapter 8: Use of High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) to Quantify Modified Nucleosides
1 Introduction
2 Materials
2.1 Setup of HPLC-MS/MS and Optimization of Nucleoside Standards
2.1.1 HPLC-MS/MS
2.1.2 Validating with Standard Curve
2.2 ALKBH3-Mediated Demethylation Assay
2.2.1 Demethylation Assay
2.2.2 Nucleic Acid Digestion
2.2.3 Quantitative Data Analysis
2.3 Quantification of N6-Methyladenosine from RNA
2.3.1 RNA Extraction
2.4 Analysis of DNA Methylation Adduct Repair in Cultured Cells
2.4.1 Induction of Methylation Damage
2.4.2 DNA Extraction and Purification
2.4.3 DNA Digestion
3 Methods
3.1 Optimization of a Standard
3.1.1 Using Optimizer
3.1.2 Validating with Standard Curve
3.2 ALKBH3-Mediated Demethylation Assay
3.2.1 Demethylation Assay
3.2.2 Nucleic Acid Digestion
3.2.3 HPLC-MS
3.2.4 Quantitative Data Analysis
3.3 Quantification of m6A in Total RNA
3.3.1 Nucleic Acid Extraction, Digestion, and Analysis
3.4 Analysis of DNA Methylation Adduct Repair in Cultured Cells
3.4.1 Induction of Methylation Damage
3.4.2 DNA Extraction and Purification
3.4.3 DNA Digestion and HPLC-MS/MS
3.4.4 Data Analysis
4 Notes
References
Chapter 9: Targeted Formation of 8-Oxoguanine in Telomeres
1 Introduction
2 Materials
2.1 Generating FAP-mCER-TRF1 Expressing Cell Lines by Lentiviral Transduction and Single Cell Cloning
2.2 Screening and Verification of FAP-mCER-TRF1 Expression and Localization by Fluorescence Microscopy
2.3 IF-FISH
2.4 FAP-Bound MG-Ester Imaging
2.5 Inducing Telomeric 8oxoG Formation
2.6 Genomic DNA Preparation for 8oxoG Detection
2.7 Enzymatic Treatments for 8oxoG Detection
2.8 S Blot Analysis of Cleaved Telomere Restriction Fragments
3 Methods
3.1 Generating FAP-mCer-TRF1 Expressing Cell Lines by Lentiviral Transduction
3.2 Propagating FAP-mCER-TRF1 Expressing Cells and Single Cell Cloning
3.3 Initial Screening of Clones with mCerulean Imaging
3.4 Verifications of FAP-mCER-TRF1 Expression and Localization
3.4.1 Verification by IF-FISH
3.4.2 Verification by FAP-Bound MG-Ester Imaging
3.5 Inducing and Verifying 8oxoG Formation at Telomeres
3.5.1 Inducing Telomeric 8oxoG Formation
3.5.2 Genomic DNA Preparation
3.5.3 Enzymatic Treatments for 8oxoG Detection
3.5.4 S Blot Analysis of Cleaved Telomere Restriction Fragments
3.5.5 Analysis of S Blot
4 Notes
References
Chapter 10: Qualitative and Quantitative Analysis of DNA Cytidine Deaminase Activity
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Qualitative Deaminase Reaction
2.3 Running Buffer and Urea-Acrylamide Gel
2.4 Quantitative Deaminase Reaction
2.5 Additional Equipment
3 Methods
3.1 Lysis
3.2 Qualitative Deaminase Reactions
3.3 Qualitative Assay Gel Electrophoresis
3.4 Quantitative Deaminase Reactions
3.5 Quantitative Deaminase Fluorescence Measurements
4 Notes
References
Chapter 11: Characterization of DNA-PK-Bound End Fragments Using GLASS-ChIP
1 Introduction
2 Materials
2.1 Cell Culture Materials (Here Specified for Standard Growth of U2OS)
3 Methods (Fig. 2)
3.1 Cell Growth
3.2 Harvesting Cells
3.3 GLASS-ChIP
3.3.1 Size Selection
3.4 Quantitation of GLASS-ChIP DNA by qPCR
4 Notes
References
Chapter 12: Monitoring Nuclease Activity by X-Ray Scattering Interferometry Using Gold Nanoparticle-Conjugated DNA
1 Introduction
2 Materials
2.1 Preparation of BSPP Protected Au Nanoparticles Via BSPP-Citrate Exchange (Au-BSPP)
2.2 Au-ssDNA Conjugation, Anion Exchange Chromatography, and Au-dsDNA Annealing
2.3 Protein Expression and Purification
2.4 DNA Substrate Preparation for the Fluorescence-Based Nuclease Reaction
2.5 Fluorescence-Based Nuclease Reaction to Validate Substrates and the Activity
2.6 Sample Preparation of XSI Experiments
3 Methods
3.1 Preparation of BSPP Protected Au Nanoparticles Via BSPP-Citrate Exchange (Au-BSPP)
3.2 Au-ssDNA Conjugation, Anion Exchange Chromatography, and Au-dsDNA Annealing
3.3 Protein Expression and Purification
3.4 DNA Substrate Preparation for the Fluorescence-Based Nuclease Reaction
3.5 Fluorescence-Based Nuclease Reaction to Validate Substrates and the Activity (Fig. 2)
3.6 Sample Preparation of XSI Experiments
3.7 XSI Data Collection at the SIBYLS Beamline (See Note 12)
3.8 Setting up XSI Data Processing Pipeline
3.9 XSI Data Analysis and Interpretation
4 Notes
References
Chapter 13: In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing
1 Introduction
2 Materials
2.1 Purification of the BRCA1-BARD1 Complex
2.1.1 Generating Recombinant Bacmid of BRCA1 and BARD1
2.1.2 Producing the Recombinant Baculovirus of BRCA1 and BARD1
2.1.3 Expression and Purification of BRCA1-BARD1 in Hi5 Cells
2.2 RAD51 Purification
2.3 DNA Preparation
2.3.1 Oligo DNA Preparation
2.3.2 Preparation of pBluescript SK II + Replicative Form I DNA and Linear pUC19 Plasmid
2.4 D-Loop Formation with RAD51 and BRCA1-BARD1
2.5 Synaptic Complex Formation Assay
3 Methods
3.1 Purification of the BRCA1-BARD1 Complex
3.1.1 Generation of BRCA1 and BARD1 Bacmid
3.1.2 BRCA1 and BARD1 Bacmid Transfection and Virus Amplification
3.1.3 Expression and Purification of BRCA1-BARD1 in Hi5 Cells
3.2 DNA Substrate Preparation
3.2.1 Purification of Oligonucleotides
3.2.2 Preparation of pBluescript SK II + Replicative Form I DNA and Linear pUC19 Plasmid
3.3 D-Loop Formation with RAD51 and BRCA1-BARD1
3.4 Synaptic Complex Formation Assay
4 Notes
References
Chapter 14: Purification of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) from HeLa Cells
1 Introduction
2 Materials
2.1 Purification of DNA-PKcs and Ku70/80 from HeLa Cells
2.2 SDS PAGE and Western Blotting of DNA-PKcs
3 Methods
3.1 Purification of DNA-PKcs and Ku70/80 from HeLa Cells
3.2 Method for SDS PAGE and Western Blot of DNA-PKcs
4 Notes
References
Chapter 15: Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells
1 Introduction
2 Materials
2.1 Expression of LigIIIα Complexes in Insect Cells
2.2 Purification of LigIIIα Protein Complexes
2.2.1 Buffers
2.2.2 ÄKTA FPLC and Columns
2.2.3 Other Equipment
2.3 Characterization of LigIIIα Protein Complexes
2.3.1 SEC-MALS
2.3.2 NS-EM
3 Methods
3.1 Expression of LigIIIα Complexes in Insect Cells
3.1.1 Transposition
3.1.2 Isolation of Recombinant Bacmid DNA
3.1.3 Transfection of Sf9 Insect Cells with Recombinant Bacmid DNA
3.1.4 Baculovirus Amplification and Screening
3.1.5 Scaled-Up Baculovirus Production in Suspension Cultures
3.1.6 Co-expression of LigIIIα, XRCC1, and TDP1 in Suspension Insect Cells by Co-infection with Two or Three Baculoviruses
3.1.7 Affinity Co-purification of LigIII Protein Complexes
3.1.8 Scaled-Up Co-expression in Insect Cells
3.2 Purification of LigIIIα Protein Complexes
3.2.1 Lysate Preparation
3.2.2 Nickel Column FPLC Using HisTrap HP Column
3.2.3 Size Exclusion Column FPLC Using HiLoad Superdex 200 16/60
3.2.4 dsDNA Cellulose Column FPLC
3.2.5 Ion-Exchange Column FPLC (HiTrap Q or HiTrap SP)
3.3 Characterization of LigIIIα Protein Complexes
3.3.1 Characterization by SEC-MALS
Column and System Equilibration in Running Buffer
Calibration Using a Known Protein and Obtaining the Molar Mass of a Sample
3.3.2 Characterization by Negative Stain Electron Microscopy
Mild Chemical Crosslinking of DNA Ligase III Samples
Negative Stain Electron Microscopy
4 Notes
References
Chapter 16: Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies
1 Introduction
2 Materials
2.1 Expressing and Purifying Ubiquitin from E. coli Cells
2.2 Expressing and Purifying the E1 Ubiquitin-Activating Enzyme UBE1 from E. coli Cells
2.3 Expressing and Purifying the S. cerevisiae Ubc13/Mms2 E2 Ubiquitin-Conjugating Enzyme Complex from E. coli Cells
2.4 Assembling and Purifying K63-Linked Polyubiquitin Chains
2.5 Pull-Down Assay with His-Tagged Ubiquitin-Binding Protein
3 Methods
3.1 Expressing and Purifying Monoubiquitin from E. coli Cells
3.2 Expressing and Purifying the E1 Ubiquitin-Activating Enzyme UBE1 from E. coli Cells
3.3 Expressing and Purifying the S. cerevisiae Ubc13/Mms2 E2 Ubiquitin-Conjugating Enzyme Complex from E. coli Cells
3.4 Assembling and Purifying K63-Linked Polyubiquitin Chains
3.5 Pull-Down Assay with His-Tagged Ubiquitin-Binding Protein
4 Notes
References
Correction to: Approaches to Monitor Termination of DNA Replication Using Xenopus Egg Extracts
Index