Base Excision Repair Pathway Methods and Protocols Methods in Molecular Biology 2701 2023rd edition by Kishor Bhakat, Tapas Hazra ISBN 9781071633724 1071633724

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Product details:

ISBN 10: 1071633724
ISBN 13: 9781071633724
Author:  Kishor K. Bhakat, Tapas K. Hazra 

This detailed volume provides a comprehensive set of experimental protocols and useful strategies to examine the repair of damaged bases via the Base Excision Repair (BER) pathway in vitro and in cells. Beginning with multiple molecular and cellular techniques to examine the excision of damaged bases from double-stranded DNA or DNA wrapped in a nucleosome, the book continues with sections covering procedures to detect and quantify the damaged bases, protein DNA crosslinks, and double-strand breaks, experimental procedures to identify DNA repair protein interactome by conventional tandem affinity purification followed by mass spectroscopy analysis, as well as the analysis of genome-wide binding of DNA repair proteins and copy number variations of the DNA damage response gene in tumors. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Table of contents:

Base Excision Repair Assays In Vitro and in Live Cells

• Simultaneous Short- and Long-Patch Base Excision Repair (BER) Assay in Live Mammalian Cells
• In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site
• Highly Sensitive Radioactivity-Based DNA 3′-Phosphatase Activity Assay for Polynucleotide Kinase 3′-Phosphatase
• Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage
• A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Excision Repair Study
• In Vitro Reconstitutive Base Excision Repair (BER) Assay

Detection and Quantification of Base Lesions, DNA Double-Strand Breaks, DNA Protein Cross-Links, and R Loops

• Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR
• Isolation and Immunodetection of Enzymatic DNA–Protein Crosslinks by RADAR Assay
• Slot Blot Assay for Detection of R Loops
• Assays with Patient-Derived Organoids to Evaluate the Impact of Microbial Infection on Base Excision Repair (BER) Enzymes
• Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods

Interactome Profiling and Purification of DNA Damage Repair/Response Proteins

• Interactome Profiling of DNA Damage Response (DDR) Mediators with Immunoprecipitation-Mass Spectrometry
• Using Affinity Pulldown Assays to Study Protein–Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9–RAD1–HUS1 (9-1-1) Complex
• Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins

Analysis of Genome-Wide Binding of DNA Repair Proteins and Copy Number Variations of DNA Damage Response Gene in Tumor

• Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues
• Genome-Wide Binding Analysis of DNA Repair Protein APE1 in Tumor Cells by ChIP-Seq
• Tumorsphere Formation Assay: A Cancer Stem-Like Cell Characterization in Pediatric Brain Cancer Medulloblastoma

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