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Substrate interactions and promiscuity in a viral DNA packaging motor

Nature volume 461, pages 669–673 (2009)Cite this article

Abstract

The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life1. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages2. Although their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage ϕ29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring5 of gene product 16 (gp16), involves two phases—an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5′–3′ strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase–substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1.

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Figure 1: Packaging of neutral DNA analogues.
Figure 2: High-resolution dynamics at a neutral-DNA insert.
Figure 3: Substrate promiscuity.
Figure 4: The motor–DNA contacts.

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Acknowledgements

We thank C. L. Hetherington, M. Kopaczynska, A. Spakowitz and J. M. Berger for critical discussions, and D. Reid, M. T. Couvillon and N. L. S. Chavez for preliminary work leading to this publication. K.A. acknowledges the PMMB fellowship through the Burroughs Wellcome Fund, A.T.P. the NIH Molecular Biophysics Training Grant, A.K. the Human Frontier Science Program Cross-Disciplinary Fellowship, J.R.M. the NSF Graduate Research Fellowship, and Y.R.C. the Burroughs Wellcome Fund Career Award at the Scientific Interface for funding. This research was supported in part by the National Institutes of Health (NIH) grants GM-071552, DE-003606 and GM-059604. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author Contributions K.A., A.T.P., A.K. and J.R.M. conducted the experiments; K.A., A.T.P., A.K., J.R.M. and Y.R.C. performed the analysis; S.G., P.J.J. and D.L.A. prepared and provided experimental materials; and K.A., A.T.P., A.K., J.R.M., Y.R.C., S.G., P.J.J. and C.B. wrote the paper. K.A., A.T.P., A.K. and J.R.M. contributed equally to this work.

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Author notes

  1. K. Aathavan & Yann R. Chemla

    Present address: Present addresses: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA (K.A.); Department of Physics and Center for Biophysics and Computational Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA (Y.R.C.).,

  2. K. Aathavan, Adam T. Politzer, Ariel Kaplan and Jeffrey R. Moffitt: These authors contributed equally to this work.

Authors and Affiliations

  1. Biophysics Graduate Group,,

    K. Aathavan, Adam T. Politzer & Carlos Bustamante

  2. Jason L. Choy Laboratory of Single-Molecule Biophysics,,

    K. Aathavan, Adam T. Politzer, Ariel Kaplan, Jeffrey R. Moffitt, Yann R. Chemla & Carlos Bustamante

  3. QB3 Institute, and,,

    Ariel Kaplan & Carlos Bustamante

  4. Department of Physics, University of California, Berkeley, California 94720, USA,

    Ariel Kaplan, Jeffrey R. Moffitt, Yann R. Chemla & Carlos Bustamante

  5. Department of Diagnostic and Biological Sciences and Institute for Molecular Virology,,

    Shelley Grimes, Paul J. Jardine & Dwight L. Anderson

  6. Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA,

    Dwight L. Anderson

  7. Departments of Molecular and Cell Biology, Chemistry, and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA,

    Carlos Bustamante

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Aathavan, K., Politzer, A., Kaplan, A. et al. Substrate interactions and promiscuity in a viral DNA packaging motor. Nature 461, 669–673 (2009). https://doi.org/10.1038/nature08443

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