Nuclease Methods and Protocols

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.

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.