Nuclease Methods and Protocols

ISBN-13: 9781617371301 / Angielski / Miękka / 2010 / 525 str.

Catherine H. Schein

Opis

Nucleases, enzymes that restructure or degrade nucleic acid polymers, are vital to the control of every area of metabolism. They range from housekeeping enzymes with broad substrate ranges to extremely specific tools (1). Many types of nucleases are used in lab protocols, and their commercial and clinical uses are expanding. The purpose of Nuclease Methods and Protocols is to introduce the reader to some we- characterized protein nucleases, and the methods used to determine their activity, structure, interaction with other molecules, and physiological role. Each chapter begins with a mini-review on a specific nuclease or a nuclease-related theme. Although many chapters cover several topics, they were arbitrarily divided into five parts: Part I, Characterizing Nuclease Activity, includes protocols and assays to determine general (processive, distributive) or specific mechanisms. Methods to assay nuclease products, identify cloned nucleases, and determine their physiological role are also included here. Part II, Inhibitors and Activators of Nucleases, summarizes assays for measuring the effects of other proteins and small molecules. Many of these inhibitors have clinical relevance. Part III, Relating Nuclease Structure and Function, provides an overview of methods to determine or model the 3-D structure of nucleases and their complexes with substrates and inhibitors. A 3-D structure can greatly aid the rational design of nucleases and inhibitors for specific purposes. Part IV, Nucleases in the Clinic, summarizes assays and protocols suitable for use with t- sues and for nuclease based therapeutics."

Spis treści

Part I. Characterizing Nuclease Activity Processivity of DNA Repair Enzymes R. Stephen Lloyd Analysis by HPLC of Distributive Activities and the Synthetic (Back) Reaction of Pancreatic-Type Ribonucleases M. Victòria Nogués and Claudi M. Cuchillo. lre1p: A Kinase and Site-Specific Endoribonuclease Tania N. Gonzalez and Peter Walter Microtiter-Plate Assay and Related Assays for Nonspecific Endonucleases Gregor Meiss, Oleg Gimadutdinow, Peter Friedhoff, and Alfred M. Pingoud Quantitating mRNAs with Relative and Competitive RT-PCR, Ellen A. Prediger Expressing Self-Incompatibility RNases (S-RNases) in Transgenic Plants Brian Beecher and Bruce A. McClure Molecular Cloning, Tissue Distribution, and Chromosomal Localization of the Human Homolog of the R2/Th/Stylar Ribonuclease Gene Family Francesco Acquati, Cinzia Nucci, Marco G. Bianchi, Tatiana Gorletta, and Roberto Taramelli Part II. Inhibitors and Activators of Nucleases Ribonuclease Inhibitors Brittan L. Pasloske Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon-g In Vitro Catherine H. Schein Assays for the Evaluation of HIV-1 Integrase Inhibitors Zeger Debyser, Peter Cherepanov, Wim Pluymers, and Erik De Clercq The Direction of Ribonucleases H by Antisense Oligodeoxynucleotides Richard V. Giles and David M. Tidd The 2-5A/RNase L Pathway and Inhibition by RNase L Inhibitor (RLI) Catherine Bisbal, Tamim Salehzada, Michelle Silhol, Camille Martinand, Florence Le Roy, and Bernard Lebleu Part III. Relating Nuclease Structure and Function Crystallization and Crystal Structure Determination of Ribonuclease A-Ribonuclease Inhibitor Protein Complex Bostjan Kobe Methods for Studying the Interaction of Barnase with Its Inhibitor Barstar GideonSchreiber Assaying In Vitro Refolding of RNases by Mass Spectrometry Gennaro Marino, Piero Pucci, and Margherita Ruoppolo Dissecting Nucleases into Their Structural and Functional Domains: Mapping the RNA-Binding Surface of RNase III by NMR Andres Ramos and Annalisa Pastore Using Electrostatics to Define the Active Site of Serratia Endonuclease Kurt L. Krause and Mitchell D. Miller Homology Modeling and Simulations of Nuclease Structures Kizhake V. Soman, Catherine H. Schein, Hongyao Zhu, and Werner Braun Methods for Determining Activity and Specificity of DNA Binding and DNA Cleavage by Class II Restriction Endonucleases Albert Jeltsch and Alfred M. Pingoud Engineered Properties and Assays for Human DNase I Mutants Clark Q. Pan, Dominick V. Sinicropi, and Robert A. Lazarus Part IV. Nucleases in the Clinic Assays for Human DNase I Activity in Biological Matrices Dominick V. Sinicropi and Robert A. Lazarus Evaluation of Retroviral Ribonuclease H Activity Jennifer T. Miller, Jason W. Rausch, and Stuart F. J. Le Grice Assays for Detection of RNase A Superfamily Ribonucleases Helene F. Rosenberg and Joseph B. Domachowske Assay for Antitumor and Lectin Activity in RNase Homologs Kazuo Nitta Isolation and Enzymatic Activity of Angiogenin James F. Riordan and Robert Shapiro Preparation and Preclinical Characterization of RNase-Based Immunofusion Proteins Dianne L. Newton and Susanna M. Rybak Part V. Assays Using Nucleases Restriction Endonucleases and Their Uses Raymond J. Williams Isolation and Characterization of an Unknown Restriction Endonuclease Raymond J. Williams Gene Modification with Hapaxoterministic Restriction Enzymes: Easing the Way, Shelby L. Berger Techniques to Measure Nucleic Acid-Protein Binding a

Blurb

Nucleases, the enzymes that cleave and shape nucleic acids, are key regulators of cellular metabolism. Angiogenesis and the unfolded protein response are just a few instances of important intra- and extracellular pathways regulated by nucleases. In Nuclease Methods and Protocols, researchers at the forefront of academic research and pharmaceutical drug development from four continents summarize their recent results. Their projects involve nucleases in many different areas of medicine and biology, ranging from protein structure and folding, to DNA repair, to developing cures for insidious diseases such as cystic fibrosis, AIDS, and cancer. Each chapter contains a minireview of a specific nuclease or nuclease-related theme, a discussion of why and when to use the assays described, and often, an explanation of how the assay was developed. The authors describe their most valuable methods for determining the mode of action, structure, interaction with other molecules, and physiological role of nucleases. Detailed step-by-step instructions and notes on avoiding pitfalls help ensure readily reproducible results.
Comprehensive and highly practical, Nuclease Methods and Protocols offers
both novice and experienced researchers a deeper understanding of the importance of nucleases in cell metabolism, growth control mechanisms, and, increasingly, in
diagnostics and therapeutics.