Poly(ADP-Ribose) Polymerase (2^nd Ed., Softcover reprint of the original 2nd ed. 2017) Methods and Protocols Methods in Molecular Biology Series, Vol. 1608

This book presents multiple new and classical methods for studying the vital poly-ADP-ribose (pADPr) pathway. Beginning with techniques for the detection and quantification of the product of poly(ADP-ribose) polymerase (PARP) enzymatic activity and detection of variation in pADPr production during the cell cycle, the volume continues with sections on the identification of pADPr protein acceptors, methods focusing on studying molecular mechanisms of PARP functions in eukaryotic cells, particularly those involved in control of DNA repair and oxidative stress, as well as in expression regulation, approaches to the in vitro reconstitution of PARP-1 interaction with chromatin, the development and testing of small molecule PARP inhibitors, and the functions of understudied members of PARP family.  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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Part I: Detection and Quantification of pADPr

 

1. Quantitation of Poly(ADP-ribose) by Isotope Dilution Mass Spectrometry

            Tabea Zubel, Rita Martello, Alexander Bürkle, and Aswin Mangerich

 

2. Quantification of PARP Activity in Human Tissues: Ex Vivo Assays in Blood Cells and Immunohistochemistry in Human Biopsies

            Eszter M. Horvath, Zsuzsanna K. Zsengellér, and Csaba Szabo

 

3. Detecting and Quantifying pADPr In Vivo

            Yi-Chen Lai, Rajesh K. Aneja, Margaret A. Satchell, and Robert S.B. Clark

 

4. Compartment-Specific Poly-ADP-Ribose Formation as a Biosensor for Subcellular NAD Pools

            Magali R. VanLinden, Marc Niere, Andrey A. Nikiforov, Mathias Ziegler, and Christian Dölle

 

5. Cell Cycle Resolved Measurements of Poly(ADP-ribose) Formation and DNA Damage Signaling by Quantitative Image-Based Cytometry

            Jone Michelena and Matthias Altmeyer

 

Part II: Identification of Protein Targets

 

6. Detecting Protein ADP-Ribosylation Using a Clickable Aminooxy Probe

            Rory K. Morgan and Michael S. Cohen

 

7. ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method

            Casey M. Daniels, Shao-En Ong, and Anthony K.L. Leung

 

8. Using Clickable NAD⁺ Analogues to Label Substrate Proteins of PARPs

            Lu Zhang and Hening Lin

 

9. Identification of Protein Substrates of Specific PARP Enzymes Using Analog-Sensitive PARP Mutants and a ‘Clickable’ NAD⁺ Analog

            Bryan A. Gibson and W. Lee Kraus

 

10. Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography – Tandem Mass Spectrometry

            Mario Leutert, Vera Bilan, Peter Gehrig, and Michael O. Hottiger

 

11. Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography –Tandem Mass Spectrometry

            Sara C. Larsen, Mario Leutert, Vera Bilan, Rita Martello, Stephanie Jungmichel, Clifford Young, Michael O. Hottiger, and Michael L. Nielsen

 

Part III: Functional Analysis

 

12. Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair

            Théo Lebeaupin, Rebecca Smith, Sébastien Huet, and Gyula Timinszky

 

13. Methods to Assess the Role of Poly(ADP-Ribose) Polymerases in Regulating Mitochondrial Oxidative Function

            Edit Mikó, Tünde Kovács, Tamás Fodor, and Péter Bai

 

14. Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV-Crosslinking and Luciferase Reporter Assay

            Yingbiao Ji

 

15. Methodology to Identify Poly-ADP-Ribose Polymerase 1 (PARP1) – mRNA Targets by PAR-CLiP

            Manana Mekishvilli, Elena Matveeva, and Yvonne Fondufe-Mittendorf

 

Part IV: Regulation of PARP-1 and Its Partners

 

16. Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

            Maggie H. Chassé, Uma M. Muthurajan, Nicholas J. Clark, Michael A. Kramer, Srinivas Chakravarthy, Thomas Irving, and Karolin Luger

 

17. PARP-1 Interaction with and Activation by Histones and Nucleosomes

            Colin Thomas, Elena Kotova, and Alexei V. Tulin

 

Part V: Designing and Testing PARP-1 Inhibitors

 

18. Strategies Employed for the Development of PARP Inhibitors

            Stacie Canan, Karen Maegley, and Nicola J. Curtin

 

19. High-Throughput Colorimetric Assay for Identifying PARP-1 Inhibitors Using a Large Small-Molecule Collection

            Elena Kotova and Alexei V. Tulin

 

20. Testing PARP Inhibitors Using a Subcutaneous Murine Xenograft Model

            Peter Makhov, Sei Naito, and Vladimir M. Kolenko

 

21. In Vitro Long Term Proliferation Assays to Study Antiproliferative Effects of PARP Inhibitors on Cancer Cells

            Heike Keilhack and Paul Chang

 

22. Use of Inosine Monophosphate Dehydrogenase Activity Assay to Determine the Specificity of PARP-1 Inhibitors

            Sajitha Anthony, Jeffrey R. Peterson, and Yingbiao Ji

 

23. The Use of PARP Inhibitors in Cancer Therapy: Use as Adjuvant with Chemotherapy or Radiotherapy, Use as a Single Agent in Susceptible Patients, and Techniques Used to Identify Susceptible Patients

            Sydney Shall, Terry Gaymes, Farzin Farzaneh, Nicola Curtin, and Ghulam J. Mufti

 

Part VI: Targeting the Understudied Components of pADPr Pathway

 

24. Purification of Recombinant Human PARP-3

            Jean-Christophe Amé, Barbara Camuzeaux, Françoise Dantzer, and Valérie Schreiber

 

25. Purification of Recombinant Human PARG and Activity Assays

            Jean-Christophe Amé, Éléa Héberlé, Barbara Camuzeaux, Françoise Dantzer, and Valérie Schreiber

 

26. Studying Catabolism of Protein ADP-Ribosylation

            Luca Palazzo, Dominic I. James, Ian D. Waddell, and Ivan Ahel

 

27. Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis

            Marie-France Langelier, Jamin D. Steffen, Amanda A. Riccio, Michael McCauley, and John M. Pascal

 

28. Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach

            Katie Pollock, Michael Ranes, Ian Collins, and Sebastian Guettler

 

29. Computational and Experimental Studies of ADP-Ribosylation

            Robert G. Hammond, Xuan Tan, Matthew Chan, Anupam Goel, and Margaret A. Johnson

Includes updated, cutting-edge methods and protocols for the study of PARP research