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Backbone tracking by the SF2 helicase NPH-II

Nature Structural & Molecular Biology volume 11, pages 526–530 (2004)Cite this article

Abstract

Members of the DExH/D family of proteins, a subset of helicase superfamily 2 (SF2), are involved in virtually all aspects of RNA metabolism. NPH-II, a prototypical member of this protein family, exhibits robust RNA helicase activity in vitro. Using a series of modified substrates to explore the unwinding mechanism of NPH-II, we observed that the helicase tracks exclusively on the loading strand, where it requires covalent continuity and specifically recognizes the ribose-phosphate backbone. NPH-II unwinding was unaffected by lesions and nicks on the top strand, which has a minimal role in substrate recognition. NPH-II required physical continuity of phosphodiester linkages on the loading strand, although abasic regions were tolerated. These findings suggest that SF2 helicases are mechanistically distinct from other helicase families that can tolerate breaks in the loading strand and for which bases are the primary recognition determinant.

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Figure 1: Probing the role of strand continuity.
Figure 2: Effects of backbone substitutions: polyglycol modifications.
Figure 3: Effects of backbone substitutions: abasic modifications.
Figure 4: Tracking by a molecular wire stripper.

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Acknowledgements

We thank Q. Yang (Case Western Reserve University) for his kind help with the experiments shown in Figure 2. We also thank the members of the Pyle lab, particularly O. Fedorova, C. Duarte, A. deLencastre and V. Serebrov, for many insightful discussions and O. Fedorova for the synthesis of oligonucleotides containing abasic clusters. We thank S. Shuman (Sloan-Kettering Institute) and C. Gross (Vertex Pharmaceuticals) for NPH-II plasmids and baculovirus constructs, protein expression protocols and many thoughtful discussions. We credit D. Wigley for the term “molecular wire stripper,” which he has used in lectures, and which we cite with his permission. We also thank S. Lomvardas (Columbia University) for help with protein expression and purification and for helpful discussions. This work was supported by a grant from the US National Institutes of Health (GMRO1 60620 to A.M.P.). A.M.P. is an investigator of the Howard Hughes Medical Institute.

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  1. Jane Kawaoka & Anna Marie Pyle

    Present address: Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, Bass Building Room 334, New Haven, Connecticut, 06520, USA

Authors and Affiliations

  1. Department of Pathology, Columbia University, New York, 10032, New York, USA

    Jane Kawaoka

  2. Departments of Biochemistry and Molecular Biophysics, Columbia University, New York, 10032, New York, USA

    Anna Marie Pyle

  3. Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, 20815, Maryland, USA

    Anna Marie Pyle

  4. Department of Biochemistry, Case Western Reserve University, Cleveland, 44106, Ohio, USA

    Eckhard Jankowsky

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Correspondence to Anna Marie Pyle.

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Kawaoka, J., Jankowsky, E. & Pyle, A. Backbone tracking by the SF2 helicase NPH-II. Nat Struct Mol Biol 11, 526–530 (2004). https://doi.org/10.1038/nsmb771

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