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Nucleic Acids Research 2005 33(2):644-649; doi:10.1093/nar/gki208
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Published online 28 January 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II

Jane Kawaoka1,2 and Anna Marie Pyle2,3,*

1 Department of Pathology, Columbia University New York, NY, USA 2 Department of Molecular Biophysics and Biochemistry, Yale University New Haven, CT, USA 3 Howard Hughes Medical Institute

*To whom correspondence should be addressed at 266 Whitney Avenue, Bass Building, Room 334, Yale University, New Haven, CT 06520, USA. Tel: +1 203 432 5633; Fax: +1 203 432 5316; Email: anna.pyle{at}yale.edu

Received October 5, 2004. Revised December 23, 2004. Accepted January 5, 2005.

NPH-II is a prototypical member of the DExH/D subgroup of superfamily II helicases. It exhibits robust RNA helicase activity, and a detailed kinetic framework for unwinding has been established. However, like most SF2 helicases, there is little known about its mode of substrate recognition and its ability to differentiate between RNA and DNA substrates. Here, we employ a series of chimeric RNA–DNA substrates to explore the molecular determinants for NPH-II specificity on RNA and to determine if there are conditions under which DNA is a substrate. We show that efficient RNA helicase activity depends exclusively on ribose moieties in the loading strand and in a specific section of the 3'-overhang. However, we also document the presence of trace activity on DNA polymers, showing that DNA can be unwound under extremely permissive conditions that favor electrostatic binding. Thus, while polymer-specific SF2 helicases control substrate recognition through specific interactions with the loading strand, alternative specificities can arise under appropriate reaction conditions.


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