This detailed volume explores poly(ADP-ribose) polymerases (PARPs) in the biology of eukaryotes and their relevance to human health. Beginning with a section on the detection and quantification of poly(ADP-ribose) polymer (pADPr), the book continues by delving into the identification of protein targets, functional analysis, the poly(ADP-ribosyl)ating pathway in chromatin and genes expression, as well as the use of animal models and PARP1 inhibitor design and testing, and more. Written for the highly successfulMethods in Molecular Biologyseries, 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 up-to-date,Poly(ADP-Ribose) Polymerase: Methods and Protocols, Third Editionpresents essential new and classical methods for studying the pADPr-pathway.

Part I: Detection and Quantification of pADPr

 

1. Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High Performance Liquid Chromatography, and Capillary Electrophoresis

            Mohsen Badiee, Audrey Boutonnet, Dat Phan, and Anthony K.L. Leung

 

2. Detecting and Quantifying pADPr In Vivo

            Andrew M. Lamade, Yaming Chen, Carla J. Johnson, Hülya Bayır, and Robert S.B. Clark

 

3. Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage

            Christopher A. Koczor, Kate M. Saville, Rasha Q. Al-Rahahleh, Joel F. Andrews, Jianfeng Li, and Robert W. Sobol

 

4. Analyzing PARP1 Activity: Small Molecule Reactants and Attached Chains of Poly(ADP-Ribose)

            Johannes Rudolph and Karolin Luger

 

5. Detecting Poly(ADP-Ribose) In Vitro and in Cells Using PAR-Trackers

            Sridevi Challa, Amy L. Whitaker, and W. Lee Kraus

 

6. An Enzyme-Linked Immunosorbent Assay to Quantify Poly(ADP-Ribose) Level In Vivo

            Chieri Ida, Sachiko Yamashita, Takayuki Eguchi, Yasuhito Kuroda, Setsu Nakae, Yoshisuke Nishi, Kazuo Kamemura, Tsuyoshi Shirai, Tamio Mizukami, Masakazu Tanaka, Joel Moss, and Masanao Miwa

 

7. Subcellular Quantitation of ADP-Ribosylation by High Content Microscopy

            Lukas Muskalla, Anka Güldenpfennig, and Michael O. Hottiger

 

8. A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery

            Johannes Gregor Matthias Rack and Ivan Ahel

 

Part II: Identification of Protein Targets

 

9. Immunoprecipitation Using Mono-ADP-Ribosylation Specific Antibodies

            Helen Dauben and Ivan Matic

 

10. A Clickable NAD⁺Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins

            Dongsheng Yao, Heba Ahmed, and Junqi Song

 

11. Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells

            Dan Huang, Andrea D. Edwards, Xuan Gong, and W. Lee Kraus

 

Part III: Functional Analysis

 

12. Studying the Immunomodulatory Functions of PARP1 and PARP2 in Mouse Models of Cancer

            Nura Lutfi, Carlos Martínez, and José Yélamos

 

13. Methods for Investigating Transient Receptor Potential Melastatin-2 (TRPM2), a Cation Channel Activated by ADP-Ribose and Involved in Cell Death

            Hannah K. Hall and David W. Koh

 

14. Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function

            Tünde Kovács, Boglárka Rauch, Edit Mikó, and Péter Bai

 

15. Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry

            Holda A. Anagho, Jonas D. Elsborg, Ivo A. Hendriks, Sara C. Buch-Larsen, and Michael L. Nielsen

 

16. Cytological Approaches to Visualize Intracellular Dynamics of RNA Binding Proteins at Active Genes in Drosophila

            Anand K. Singh and Subhash C. Lakhotia

 

Part IV: Poly(ADP-Ribosyl)ating Pathway in Chromatin and Gene Expression

 

17. Chromatin Immunoprecipitation Approach to Determine How PARP1 Domains Affect Binding Pattern to Chromatin

            Guillaume Bordet, Gbolahan Bamgbose, Sayem H. Bhuiyam, Sarah Johnson, and Alexei V. Tulin

 

18. Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates

            Hejer Dhahri, Elena Matveeva, and Yvonne Fondufe-Mittendorf

 

19. Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells

            Peter Makhov, Rushaniya Fazliyeva, Antonio Tufano, Robert G. Uzzo, and Vladimir M. Kolenko

 

Part V: Using Animal Models

 

20. Using Drosophila Genetics to Identify Factors that Affect PARP1 Activity In Vivo

            Guillaume Bordet and Alexei V. Tulin

 

21. Generating PARP KnockoutD. Melanogasterwith CRISPR/Cas9 System

            Yaroslava Karpova and Alexei V. Tulin

 

22. TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice

            Yaroslava Karpova and Alexei V. Tulin

 

Part VI: Designing and Testing PARP-1 Inhibitors

 

23. Cell-Based Screening for New PARP Inhibitors Utilizing PARG Mutated Mouse Embryonic Stem Cells

            Yaroslava Karpova, Danping Guo, and Alexei V. Tulin

 

24. Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase System

            Anna K. Duell, Daniel J. Sanderson, and Michael S. Cohen

 

Part VII: Targeting the Understudied Components of pADPr Pathway

 

25. Purification of Recombinant Human PARG and Activity Assays

            Jean-Christophe Amé and Françoise Dantzer

 

26. Purification of Recombinant Human PARP-3

            Jean-Christophe Amé, Leonel Nguekeu-Zebase, Daisy Harwood, Zuleyha Yildirim, Lisa Roegel, Agathe Boos, and Françoise Dantzer