Preface
Contents
Contributors
Part I: Base Excision Repair Assays In Vitro and in Live Cells
Chapter 1: Simultaneous Short- and Long-Patch Base Excision Repair (BER) Assay in Live Mammalian Cells
1 Introduction
2 Materials
2.1 Construction of DNA Carrying a Single Lesion (Undamaged Base/eA/8-oxoG/5-OHU)
2.2 In-Cell Repair and Patch Formation Assay
3 Methods
3.1 Construction of Covalently Closed Circular DNA Carrying a Single Lesion (Undamaged Base/eA/8-oxoG/5-OHU; See Note 3)
3.1.1 Phosphorylation of the Adduct-Containing Oligonucleotides
3.1.2 Separation of Phosphorylated Oligonucleotides from Free ATP Using MicroSpin G-25 Columns (See Notes 5-6)
3.1.3 Annealing Phosphorylated Oligo to ssDNA Template
3.1.4 Extension (See Note 10)
3.1.5 Ligation
3.1.6 Supercoil-It
3.1.7 Purification
3.2 In-Cell Repair and Patch Formation Assay
3.2.1 Transfection of Mammalian Cells with the Lesion-Containing Plasmids
3.2.2 Harvesting Plasmid DNA from Mammalian Cells
3.2.3 Restriction Enzyme Digestions
3.2.4 Transformation
Transformation Using Electrocompetent Cells
Transformation Using Chemically Competent Cells (Alternative to electroporation)
3.2.5 Plating (See Notes 23-24)
3.2.6 Counting and Calculations
4 Notes
References
Chapter 2: In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site
1 Introduction
2 Materials
2.1 Purification of Recombinant APE1 Protein (rAPE1)
2.2 Whole-Cell Extract (WCE) Preparation
2.2.1 Cell Growth
2.2.2 WCE Protein Extraction
2.3 SDS-PAGE
2.4 Oligonucleotide Substrate Preparation
2.5 APE1 Endodeoxyribonuclease Cleavage Reaction
2.6 Casting and Running of Denaturing Urea Gel for Determining rAP Cleavage
3 Methods
3.1 Purification and Absorbance Measurement of rAPE1 Concentration
3.1.1 Preparation of E. Coli Lysate Overexpressing APE1
3.1.2 GSTrap Purification of rAPE1
3.1.3 GST Tag Cleavage and HiTrap Benzamidine Purification
3.1.4 HiTrap SP FF Purification of rAPE1
3.2 WCE Preparation and Quantification
3.2.1 Harvesting of Cell Culture
3.2.2 Preparation and Quantification of WCE
3.3 Casting and Running of SDS-PAGE Gel
3.3.1 Casting of SDS-PAGE Gel
3.3.2 Running of SDS-PAGE Gel for Determining rAPE1 Concentration
3.3.3 Running of SDS-PAGE Gel for WCE
3.4 Annealing of DNA Oligonucleotides
3.4.1 Set Up the rAP Endoribonuclease Assay by Using rAPE1 or WCE
3.4.2 Dose-Response Analysis
3.4.3 Time Course Analysis
3.4.4 Specific Inhibition of APE1 Enzymatic Activity by Using Inhibitor III
3.5 Casting and Running of Urea Gel for Determining rAP Cleavage
3.6 Analysis of Results
4 Notes
References
Chapter 3: Highly Sensitive Radioactivity-Based DNA 3โฒ-Phosphatase Activity Assay for Polynucleotide Kinase 3โฒ-Phosphatase
1 Introduction
2 Materials
2.1 In Vitro Assay for PNKPยดs 3โฒ-Phosphatase Activity
2.2 Assay of In Vitro Single-Strand Break Repair Activity
3 Methods
3.1 In Vitro Assay of 3โฒ-Phosphatase Activity of PNKP
3.1.1 Labeling the 5โฒ End of U-26 Uracil-Containing Oligo with ATP ฮณ-32P
3.1.2 Annealing with F-25 and G-51 Complementary Oligos
3.1.3 Ligation (See Note 6)
3.1.4 Udg and Fpg Digestion of the Ligated Product (See Note 7)
3.1.5 Removal of the Free, Unincorporated ATP-ฮณ-32P from the Udg/Fpg-Digested Reaction Mixture
3.1.6 Checking the Purity of the Radiolabeled Substrate
3.1.7 Preparation of the Cold Substrate (for Evaluation of the Radiolabeled Product Formation in the Linear Range)
3.1.8 Measuring the Concentration of Pure Proteins
3.1.9 Assay of 3โฒ-Phosphatase Activity of PNKP
3.1.10 Quantitation of the 3โฒ-Phosphatase Activity
3.2 PNKP-Mediated Single-Strand Break Repair Activity Assay In Vitro
3.2.1 Preparation of Substrate by Annealing the Oligos
3.2.2 Measuring the Concentration of Pure Proteins (See Note 20)
3.2.3 Assay of Single-Strand Break Repair Activity In Vitro (a Schematic Representation Is Shown in Fig. 2a)
3.2.4 Quantitation of the Total Repair Activity
4 Notes
References
Chapter 4: Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage
1 Introduction
2 Materials
2.1 Instruments and Equipment
2.2 Reagents
2.3 Buffers, Solutions, and Media
3 Methods
3.1 Generation and Purification of Damaged DNA Substrates
3.1.1 Design of Damaged DNA Substrates
3.1.2 Ligation of Damaged DNA Substrates
3.1.3 Purification of Damaged DNA Substrates
3.1.4 Annealing and Storage of Damaged DNA Substrates
3.2 Histone Expression and Purification
3.2.1 Histone Plasmid Generation
3.2.2 Histone Expression
3.2.3 Histone Lysis and Extraction from Inclusion Bodies
3.2.4 Histone Purification
3.3 Generation of H2A/H2B Dimer and H3/H4 Tetramer
3.3.1 Refolding of H2A/H2B Dimer
3.3.2 Refolding of H3/H4 Tetramer
3.3.3 Purification of H2A/H2B Dimer and H3/H4 Tetramer
3.4 Reconstituting Nucleosomes Containing DNA Damage
3.4.1 Nucleosome Reconstitution
3.4.2 Nucleosome Purification
4 Notes
References
Chapter 5: A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Exc...
1 Introduction
2 Materials
2.1 Chemical Synthesis of Defined Oligonucleotide Containing a Site-Directed Lesion
2.2 The Basic Steps of Synthesis of a Modified Phosphoramidite and Its Insertion into a Defined Oligomer Involve
3 Methods
3.1 The Enzymatic Assay: Radiolabeling of Template Oligonucleotide and Annealing to Complementary Strand
3.2 Enzymatic Reaction
3.3 Detection of Oligomer Cleavage on a Denaturing PAGE
3.4 NIR and NER Assays
3.5 Primer Extension Assay with a DNA Polymerase
4 Notes
References
Chapter 6: In Vitro Reconstitutive Base Excision Repair (BER) Assay
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Laboratory Reagents
2.3 Oligonucleotides
3 Methods
3.1 Oligonucleotide Radiolabeling
3.2 Oligonucleotide Annealing
3.3 Recombinant Protein Purification
3.4 Base Excision Repair Assay Procedures
3.4.1 Nuclease Activity Assay of APE1
3.4.2 dRP Lyase Activity of DNA Polymerase ฮฒ
3.4.3 FEN1 Activity Assay
3.4.4 DNA Ligase 1 or XRCC1/Ligase 3 Activity Assay
3.4.5 In Vitro Reconstitution of SP- and LP-BER Assay
4 Notes
References
Part II: Detection and Quantification of Base Lesions, DNA Double-Stand Breaks, DNA Protein Cross-Links, and R Loops
Chapter 7: Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR
1 Introduction
1.1 Importance and Difficulties to Detect Guanine Base Lesions
1.2 Principle of the Assay
1.3 Advantages of FLARE-Coupled qPCR
1.4 Limitations of the Assay
2 Materials
3 Methods
3.1 DNA Extraction from Animal Tissues and Cultured Cells
3.2 DNA Extraction Using Qiagen Genomic-Tips Kit (Follow Recommendation of Manufacturer Provided with the Kit)
3.3 DNA Quantitation (See Note 6)
3.3.1 NanoDrop Quantification
3.3.2 PicoGreen Quantitation of DNA
3.4 Restriction Digestion of Genomic or Mitochondrial DNA
3.5 Generation of DNA Polymerase-Blocking Gaps in DNA by OGG1 (See Notes 8 and 9)
3.6 Quantitative (q) Polymerase Chain Reaction (qPCR) (See Note 10)
3.6.1 Primer Design for qPCR (Things to Consider; See Note 11)
3.6.2 Primer Solubilization (See Note 13)
3.6.3 Primer Validation
3.6.4 Experimental Steps
3.7 Data Analysis
3.8 Statistical Analysis
4 Notes
References
Chapter 8: Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay
1 Introduction
2 Materials
2.1 Cell Culture and Drug Treatment
2.2 DPC Isolation
2.3 Slot Blotting, Immunodetection, and Analysis
3 Methods
3.1 Cell Culture
3.2 Drug Treatment
3.3 Cell Lysis and Sample Preparation
3.4 DNA Quantification and Sample Dilution
3.5 Slot Blotting
3.6 Immunodetection
3.7 Data Analysis
4 Notes
References
Chapter 9: Slot Blot Assay for Detection of R Loops
1 Introduction
2 Materials
2.1 Components
2.2 Antibodies
3 Methods
3.1 Purification of Genomic DNA
3.2 Digestion of Genomic DNA with Restriction Enzyme to Reduce Viscosity
3.3 Ribonuclease Digestion to Determine Specificity of the S9.6 Antibody
3.4 Blotting of DNA Samples onto Nylon Membrane and UV Crosslink
3.5 Detection of R Loops Using S9.6 Antibody
3.6 Quantification of R Loops
4 Notes
References
Chapter 10: Assays with Patient-Derived Organoids to Evaluate the Impact of Microbial Infection on Base Excision Repair (BER) ...
1 Introduction
2 Materials
2.1 Culture of Fusobacterium nucleatum
2.2 The Development of Enteroid-Derived Monolayer (EDM)
2.3 Immunoblotting
2.4 Immunofluorescence
2.5 Functional Assays with the EDM Model
3 Methods
3.1 Culture of Fusobacterium nucleatum (Fn)
3.2 The Development and Characterization of 3D Organoids
3.3 The Development of 2D Polarized EDMs from 3D Colon Organoids
3.4 Infection of EDMs with Fn and Functional Characterization
3.5 Assessment of the Transcriptional Level of the BER Enzymes
3.6 Assessment of the Translational Level of BER Enzymes
3.7 Immunofluorescence of ฮณH2AX Phosphorylation (Fig. 3a)
3.8 Measurement of DNA/RNA Oxidative Damage by ELISA
4 Notes
References
Chapter 11: Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods
1 Introduction
2 Materials
3 Methods
3.1 Reporter Plasmid Preparation
3.1.1 Generation of a Plasmid Construct, Named pNt.Nb, Containing a Pair of Microhomology Sequences, GTGAGG and CCTCAGC, Site ...
3.1.2 Generation of the U-Containing Circular Plasmid, pNtU.NbU
3.1.3 Generation of Linearized Reporter Plasmid, pNS with 3โฒ-P and 5โฒ-P Termini
3.2 In-Cell NHEJ/Alt-EJ Repair Assay
3.2.1 Transfection of Mammalian Cells with pNS (Fig. 3)
3.2.2 Extraction of Recircularized Plasmid Using the Modified Hirt Method
3.2.3 Recircularized Plasmid Enrichment and Sequencing
3.3 In Vitro NHEJ/Alt-EJ Repair Assay
3.3.1 Nuclear Extract Preparation and Co-immunoprecipitation
3.3.2 In Vitro Plasmid Recircularization Assay
4 Notes and Final Summary
References
Part III: Interactome Profiling and Purification of DNA Damage Repair/Response Proteins
Chapter 12: Interactome Profiling of DNA Damage Response (DDR) Mediators with Immunoprecipitation-Mass Spectrometry
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Reagents
2.3 Consumables
2.4 Equipment
2.5 Nanoflow HPLC and Mass Spectrometry Analysis
2.6 Database Search
3 Methods
3.1 Induction of DDR on 293FT Cells
3.2 Dynabeads Conjugation and Immunoprecipitation
3.3 Workflow A: In-Gel Digestion
3.4 Workflow B: On-Bead Digestion
3.5 Sample Cleaning Prior to LC-MS/MS
3.6 Nanoflow HPLC and Mass Spectrometry Analysis
3.7 Database Search
4 Notes
References
Chapter 13: Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint...
1 Introduction
2 Materials
2.1 Purified Proteins
2.2 Buffers and Affinity Chromatography Resins
2.3 Equipment
3 Methods
3.1 Column Equilibration
3.1.1 Column Equilibration for Anti-Flag M2 Column
3.1.2 Column Equilibration for Ni-NTA Column
3.2 Column Batch Binding
3.3 Column Batch Washing
3.4 Column Batch Elution
3.5 SDS-PAGE Analysis
4 Notes
References
Chapter 14: Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Buffers
2.3 Consumables and Other Items
3 Methods
3.1 Growing Culture
3.2 Harvesting Cells
3.3 WCE Preparation
3.4 Incubation of WCE with IgG-Sepharose Beads
3.5 Washing of the IgG-Sepharose Beads
3.6 TEV Cleavage
3.7 Incubation of TEV Eluate with Calmodulin-Sepharose Beads
3.8 Protein Elution from Calmodulin-Sepharose Beads
3.9 Protein Quantification by Bradford Assay
3.10 Concentrating Eluted Proteins from the Calmodulin-Sepharose Beads
3.11 SDS-PAGE
3.12 Silver Staining
3.13 Mass-Spectrometric Analysis of the Interacting Proteins
4 Notes
References
Part IV: Analysis of Genome-Wide Binding of DNA Repair Proteins and Copy Number Variations of DNA Damage Response Gene in Tumor
Chapter 15: Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues
1 Introduction
1.1 Significance of Examining Copy Number Variation of Individual Genes
2 Materials
2.1 NanoString Probe Code Set
2.2 Genome DNA Preparation from FFPE Slides
2.3 AluI Digestion of the Genomic DNA
3 Methods: Analysis of CNV Using NanoString Sprint
3.1 Determination of Region of Interest (ROI) in FFPE Tissues
3.2 Genome DNA Preparation from FFPE Slides
3.2.1 Removal of Paraffin from FFPE Tissues
3.2.2 Marking ROIs on the Tissue Slides
3.2.3 Lysis
3.2.4 Confirmation of DNA
3.3 AluI Digestion of Extracted DNA
3.4 Assay Using NanoString Sprint and the Interpretation of Results
3.5 Quality Control
4 Notes
References
Chapter 16: Genome-Wide Binding Analysis of DNA Repair Protein APE1 in Tumor Cells by ChIP-Seq
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Reagents
2.3 Buffers and Solutions
3 Methods
3.1 Cell Culture and DNA Protein Cross-Linking
3.2 Checking Fragmentation Size
3.3 DNA Immunoprecipitation
3.4 Bead Washing
3.5 DNA Cleanup
3.6 ChIP Library Preparation
3.6.1 Removal of Unwanted DNA Fragments
3.6.2 End Repair
3.6.3 A-Tail Addition
3.6.4 Adapter Ligation
3.6.5 PCR Amplification
3.7 AcAPE1 ChIP-Seq Analysis
4 Notes
References
Chapter 17: Tumorsphere Formation Assay: A Cancer Stem-Like Cell Characterization in Pediatric Brain Cancer Medulloblastoma
1 Introduction
2 Materials
2.1 MB Cell Lines
2.2 Reagents
2.3 Laboratory Equipment
3 Methods
4 Notes
References
Index