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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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References

  1. Iyer, L. M., Makarova, K. S., Koonin, E. V. & Aravind, L. Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging. Nucleic Acids Res. 32, 5260–5279 (2004)

    Article  CAS  Google Scholar 

  2. Burroughs, A. M., Iyer, L. M. & Aravind, L. Comparative Genomics and Evolutionary Trajectories of Viral ATP Dependent DNA-Packaging Systems 48–65 (Basel, 2007)

    Google Scholar 

  3. Hopfner, K. P. & Michaelis, J. Mechanisms of nucleic acid translocases: lessons from structural biology and single-molecule biophysics. Curr. Opin. Struct. Biol. 17, 87–95 (2007)

    Article  CAS  Google Scholar 

  4. Grimes, S., Jardine, P. J. & Anderson, D. Bacteriophage φ29 DNA packaging. Adv. Virus Res. 58, 255–294 (2002)

    Article  CAS  Google Scholar 

  5. Morais, M. C. et al. Defining molecular and domain boundaries in the bacteriophage φ29 DNA packaging motor. Structure 16, 1267–1274 (2008)

    Article  CAS  Google Scholar 

  6. Moffitt, J. R. et al. Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446–450 (2009)

    Article  ADS  CAS  Google Scholar 

  7. Chemla, Y. R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005)

    Article  CAS  Google Scholar 

  8. Thiviyanathan, V. et al. Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and S stereochemistry. Biochemistry 41, 827–838 (2002)

    Article  CAS  Google Scholar 

  9. Strauss, J. K. & Maher, L. J. DNA bending by asymmetric phosphate neutralization. Science 266, 1829–1834 (1994)

    Article  ADS  CAS  Google Scholar 

  10. Eoff, R. L., Spurling, T. L. & Raney, K. D. Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase. Biochemistry 44, 666–674 (2005)

    Article  CAS  Google Scholar 

  11. Kawaoka, J., Jankowsky, E. & Pyle, A. M. Backbone tracking by the SF2 helicase NPH-II. Nature Struct. Biol. 11, 526–530 (2004)

    Article  CAS  Google Scholar 

  12. SenGupta, D. J. & Borowiec, J. A. Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen. Science 256, 1656–1661 (1992)

    Article  ADS  CAS  Google Scholar 

  13. Mancini, E. J. et al. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118, 743–755 (2004)

    Article  CAS  Google Scholar 

  14. Dillingham, M. S., Soultanas, P. & Wigley, D. B. Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation. Nucleic Acids Res. 27, 3310–3317 (1999)

    Article  CAS  Google Scholar 

  15. Howard, J. Mechanics of Motor Proteins and the Cytoskeleton 1st edn 62 (Sinauer Associates, 2001)

    Google Scholar 

  16. Stanley, L. K. et al. When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I. EMBO J. 25, 2230–2239 (2006)

    Article  CAS  Google Scholar 

  17. Enemark, E. J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006)

    Article  ADS  CAS  Google Scholar 

  18. Pearson, R. K. & Fox, M. S. Effects of DNA heterologies on bacteriophage lambda packaging. Genetics 118, 5–12 (1988)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Moll, W. D. & Guo, P. Translocation of nicked but not gapped DNA by the packaging motor of bacteriophage φ29. J. Mol. Biol. 351, 100–107 (2005)

    Article  CAS  Google Scholar 

  20. Oram, M., Sabanayagam, C. & Black, L. W. Modulation of the packaging reaction of bacteriophage t4 terminase by DNA structure. J. Mol. Biol. 381, 61–72 (2008)

    Article  CAS  Google Scholar 

  21. Agresti, A. Categorical Data Analysis Chs 5–6 (Wiley-Interscience, 2003)

    MATH  Google Scholar 

  22. Shen, J., Gai, D., Patrick, A., Greenleaf, W. B. & Chen, X. S. The roles of the residues on the channel β-hairpin and loop structures of simian virus 40 hexameric helicase. Proc. Natl Acad. Sci. USA 102, 11248–11253 (2005)

    Article  ADS  CAS  Google Scholar 

  23. Barkow, S. R., Levchenko, I., Baker, T. A. & Sauer, R. T. Polypeptide translocation by the AAA+ ClpXP protease machine. Chem. Biol. 16, 605–612 (2009)

    Article  CAS  Google Scholar 

  24. Mulkidjanian, A. Y., Makarova, K. S., Galperin, M. Y. & Koonin, E. V. Inventing the dynamo machine: the evolution of the F-type and V-type ATPases. Nature Rev. Microbiol. 5, 892–899 (2007)

    Article  CAS  Google Scholar 

  25. Smith, D. E. et al. The bacteriophage φ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001)

    Article  ADS  CAS  Google Scholar 

  26. Lewis, J. & Sauro, J. When 100% really isn’t 100%: improving the accuracy of small-sample estimates of completion rates. J. Usability Stud. 1, 136–150 (2006)

    Google Scholar 

  27. Agresti, A. & Coull, B. Approximate is better than “exact” for interval estimation of binomial proportions. Am. Stat. 52, 119–126 (1998)

    MathSciNet  Google Scholar 

  28. Rickgauer, J. P. et al. Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage ϕ29. Biophys. J. 94, 159–167 (2008)

    Article  ADS  CAS  Google Scholar 

  29. Smith, S. B., Cui, Y. & Bustamante, C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 361, 134–162 (2003)

    Article  CAS  Google Scholar 

  30. Moffitt, J. R., Chemla, Y. R., Izhaky, D. & Bustamante, C. Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc. Natl Acad. Sci. USA 103, 9006–9011 (2006)

    Article  ADS  CAS  Google Scholar 

Download references

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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This file contains Supplementary Tables 1-6, a Supplementary Discussion, Supplementary Data, Supplementary Figures 1-2 with Legends and Supplementary References. (PDF 596 kb)

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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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