Poly(ADP-Ribose) Polymerase

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.
Authoritative and practical, Poly(ADP-Ribose) Polymerase: Methods and Protocols, Second Edition serves as an ideal companion to the first edition for scientists whose investigations involve this important pathway.
The chapter 'Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach' is published open access under a CC BY 4.0 license.

Includes updated, cutting-edge methods and protocols for the study of PARP research Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Inhalt

Quantitation of Poly(ADP-ribose) by Isotope Dilution Mass Spectrometry.- Quantification of PARP Activity in Human Tissues: Ex Vivo Assays in Blood Cells and Immunohistochemistry in Human Biopsies.- Detecting and Quantifying pADPr In Vivo.- Compartment-Specific Poly-ADP-Ribose Formation as a Biosensor for Subcellular NAD Pools.- Cell Cycle Resolved Measurements of Poly(ADP-ribose) Formation and DNA Damage Signaling by Quantitative Image-Based Cytometry.- Detecting Protein ADP-Ribosylation Using a Clickable Aminooxy Probe.- ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method.- Using Clickable NAD + Analogues to Label Substrate Proteins of PARPs.- Identification of Protein Substrates of Specific PARP Enzymes Using Analog-Sensitive PARP Mutants and a 'Clickable' NAD + Analog.- Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography Tandem Mass Spectrometry.- Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography Tandem Mass Spectrometry.- Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair.- Methods to Assess the Role of Poly(ADP-Ribose) Polymerases in Regulating Mitochondrial Oxidative Function.- Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV-Crosslinking and Luciferase Reporter Assay.- Methodology to Identify Poly-ADP-Ribose Polymerase 1 (PARP1) mRNA Targets by PAR-CLiP.- Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin.- PARP-1 Interaction with and Activation by Histones and Nucleosomes.- Strategies Employed for the Development of PARP Inhibitors.- High-Throughput Colorimetric Assay for Identifying PARP-1 Inhibitors Using a Large Small-Molecule Collection.- Testing PARP Inhibitors Using a Subcutaneous Murine Xenograft Model.- In Vitro Long Term Proliferation Assays to Study Antiproliferative Effectsof PARP Inhibitors on Cancer Cells.- Use of Inosine Monophosphate Dehydrogenase Activity Assay to Determine the Specificity of PARP-1 Inhibitors.- 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.- Purification of Recombinant Human PARP-3.- Purification of Recombinant Human PARG and Activity Assays.- Studying Catabolism of Protein ADP-Ribosylation.- Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis.- Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach.- Computational and Experimental Studies of ADP-Ribosylation.