Nucleic Acids Research Advance Access originally published online on August 25, 2006
Nucleic Acids Research 2006 34(15):4232-4244; doi:10.1093/nar/gkl451
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Nucleic Acids Research, 2006, Vol. 34, No. 15 4232-4244
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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DNA mechanics as a tool to probe helicase and translocase activity
1 Laboratoire de Physique Statistique de l' Ecole Normale Supérieure, UMR 8550 CNRS 24 rue Lhomond, 75231 Paris Cedex 05, France 2 Département de Biologie, Ecole Normale Supérieure 46 rue d'Ulm, 75231 Paris Cedex, 05, France 3 Laboratoire Pierre Aigrain, Ecole Normale Supérieure UMR 8551 CNRS, 24 rue Lhomond, 75231 Paris Cedex 05, France 4 Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS UMR5100 Toulouse, France 5 Centre de Génétique Moléculaire, CNRS UPR2167 Gif-sur-Yvette, France
*To whom correspondence should be addressed at Laboratoire de Physique Statisque de l’ Ecole Normale Supérieure, 24 rue Lhomond, 75005 Paris, France. Tel: 33 1 44 32 34 92; Fax: 33 1 44 32 34 33; Email: Vincent.Croquette{at}lps.ens.fr
Received March 31, 2006. Revised June 6, 2006. Accepted June 12, 2006.
Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.
Present address: Omar A. Saleh, Materials Department, University of California, Santa Barbara, CA 93106, USA
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