Helicases Methods and Protocols
In recent years, much information has been revealed concerning the essential role of helicases, the enzymes that utilize the energy derived from nucleoside triphosphate hydrolysis to unwind the double stranded helical structure of nucleic acids. In Helica
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M O L E C U L A R B I O L O G Y TM
Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to www.springer.com/series/7651
Helicases Methods and Protocols
Edited by
Mohamed M. Abdelhaleem University of Toronto, Toronto, ON, Canada
Editor Mohamed M. Abdelhaleem Hospital for Sick Children Department of Paediatric Laboratory Medicine 555 University Ave. Toronto ON M5G 1X8 Canada [email protected]
ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60327-354-1 e-ISBN 978-1-60327-355-8 DOI 10.1007/978-1-60327-355-8 Library of Congress Control Number: 2009936373 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper springer.com
Preface The objective of this book is to provide the scientific community with the current methods used to study helicases, the enzymes that utilize the energy derived from nucleoside triphosphate (NTP) hydrolysis to unwind the double-stranded helical structure of nucleic acids. The book starts with an overview chapter that provides a brief introduction of helicases, with few examples of their role in nucleic acid metabolism. This chapter is intended for readers new to the field. The chapters that follow are written by leading international scientists who contributed significantly to our current understanding of helicases. In these chapters, the reader finds methods for the production and purification of helicases from different species as well as detailed studies of helicase activities, including NTP binding and hydrolysis, nucleic acid binding and unwinding, and translocation along nucleic acid substrates. Helicase activities are generally measured by methods that rely on radiometric, enzymatic, and fluorescence-based techniques. As described in Chapter 2, fluorescence probes have high sensitivity and rapid response that permit real time analysis. The ability to use fluorescence probes to investigate helicase activity at the single molecule level has provided significant insights into the mechanism of helicase function. A general guide to such assays is given in Chapter 3. Another advance in helicase studies is the use of pre-steady state kinetic techniques, as described for the translocase activ
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