Base Excision Repair Pathway

ISBN-13: 9781071633724 / Angielski / Twarda / 2023

Opis

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. 

Authoritative and practical, Base Excision Repair Pathway: Methods and Protocols serves as a valuable resource for novices and experts trying to examine the repair of various types of DNA lesions in vitro and in cell by the distinct set of proteins in the BER pathway.

Spis treści

Part I: Base Excision Repair Assays In Vitro and in Live Cells

 

1. Simultaneous Short and Long Patch Base Excision Repair (BER) Assay in Live Mammalian Cells

            Rabindra Roy

 

2. In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site

            Matilde Clarissa Malfatti, Giulia Antoniali, and Gianluca Tell

 

3. Highly Sensitive Radioactivity-Based DNA 3’-Phosphatase Activity Assay for Polynucleotide Kinase 3’-Phosphatase

            Anirban Chakraborty and Tapas Hazra

 

4. Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage

            Benjamin J. Ryan, Tyler M. Weaver, Jonah J. Spencer, and Bret D. Freudenthal

 

5. A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Excision Repair Study

            Bo Hang

 

6. In Vitro Reconstitutive Base Excision Repair (BER) Assay

            Aruna S. Jaiswal, Elizabeth A. Williamson, Arunima S. Jaiswal, Kimi Kong, and Robert A. Hromas

 

Part II: Detection and Quantification of Base Lesions, DNA Double-Stand Breaks, DNA Protein Cross-Links, and R Loops

 

7. Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR

            Pan Lang, Xue Yaoyao, Wang Ke, Zheng Xu, and Istvan Boldogh

 

8. Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay

            Megan Perry and Gargi Ghosal

 

9. Slot Blot Assay for Detection of R Loops

            Altaf H. Sarker and Priscilla K. Cooper

 

10. Assays with Patient-Derived Organoids to Evaluate the Impact of Microbial Infection on the Base Excision Repair (BER) Enzymes

            Ibrahim M. Sayed, Anirban Chakraborty, and Soumita Das

 

11. Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods

            Arijit Dutta, Joy Mitra, Pavana M. Hegde, Sankar Mitra, and Muralidhar L. Hegde

 

Part III: Interactome Profiling and Purification of DNA Damage Repair/Response Proteins

 

12. Interactome Profiling of DNA Damage Response (DDR) Mediators with Immunoprecipitation-Mass Spectrometry

            Henry C.-H. Law, Dragana Noe, and Nicholas T. Woods

 

13. Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex

            Drew T. McDonald, Pam S. Wang, Jennifer Moitoza Johnson, and Miaw-Sheue Tsai

 

14. Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins

            Amala Kaja, Priyanka Barman, Shalini Guha, and Sukesh R. Bhaumik

 

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

 

15. Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues

            Tadahide Izumi

 

16. Genome-Wide Binding Analysis of DNA Repair Protein APE1 in Tumor Cells by ChIP-Seq

            Mason Tarpley, Yingling Chen, and Kishor K. Bhakat

 

17. Tumorsphere Formation Assay: A Cancer Stem-Like Cell Characterization in Pediatric Brain Cancer Medulloblastoma

            Sutapa Ray

Blurb

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 topis, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and practical, Base Excision Repair Pathway: Methods and Protocols serves as a valuable resource for novices and experts trying to examine the repair of various types of DNA lesions in vitro and in cell by the distinct set of proteins in the BER pathway.