DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate


Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules1,2,3. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour4,5,6. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably6,7,8, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Unwinding of DNA by individual RecBCD molecules is heterogeneous, with a fixed rate for the duration of DNA translocation.
Figure 2: The DNA unwinding rate of single enzymes is stochastically changed to a velocity within the original distribution, after transient depletion of Mg2+-ATP.


  1. 1

    Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008)

    CAS  Article  Google Scholar 

  2. 2

    Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Xie, X. S. & Lu, H. P. Single-molecule enzymology. J. Biol. Chem. 274, 15967–15970 (1999)

    CAS  Article  Google Scholar 

  4. 4

    Spies, M. et al. A molecular throttle: the recombination hotspot χ controls DNA translocation by the RecBCD helicase. Cell 114, 647–654 (2003)

    CAS  Article  Google Scholar 

  5. 5

    Spies, M., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. RecBCD enzyme switches lead motor subunits in response to χ recognition. Cell 131, 694–705 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Handa, N., Bianco, P. R., Baskin, R. J. & Kowalczykowski, S. C. Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition. Mol. Cell 17, 745–750 (2005)

    CAS  Article  Google Scholar 

  7. 7

    Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Dillingham, M. S. & Kowalczykowski, S. C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008)

    CAS  Article  Google Scholar 

  9. 9

    Lam, S. T., Stahl, M. M., McMilin, K. D. & Stahl, F. W. Rec-mediated recombinational hot spot activity in bacteriophage lambda. II. A mutation which causes hot spot activity. Genetics 77, 425–433 (1974)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Dillingham, M. S., Spies, M. & Kowalczykowski, S. C. RecBCD enzyme is a bipolar DNA helicase. Nature 423, 893–897 (2003)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Taylor, A. F. & Smith, G. R. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature 423, 889–893 (2003)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Handa, N. et al. Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, χ, by RecBCD enzyme. Proc. Natl Acad. Sci. USA 109, 8901–8906 (2012)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Singleton, M. R., Dillingham, M. S., Gaudier, M., Kowalczykowski, S. C. & Wigley, D. B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Dillingham, M. S., Webb, M. R. & Kowalczykowski, S. C. Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme. J. Biol. Chem. 280, 37069–37077 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Frauenfelder, H., McMahon, B. H., Austin, R. H., Chu, K. & Groves, J. T. The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin. Proc. Natl Acad. Sci. USA 98, 2370–2374 (2001)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Lu, H. P., Xun, L. & Xie, X. S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Xue, Q. & Yeung, E. S. Differences in the chemical reactivity of individual molecules of an enzyme. Nature 373, 681–683 (1995)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Craig, D. B., Arriaga, E. A., Wong, J. C. Y., Lu, H. & Dovichi, N. J. Studies on single alkaline phosphatase molecules: reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation – the death of an enzyme. J. Am. Chem. Soc. 118, 5245–5253 (1996)

    CAS  Article  Google Scholar 

  19. 19

    Shi, J. et al. Multiple states of the Tyr318Leu mutant of dihydroorotate dehydrogenase revealed by single-molecule kinetics. J. Am. Chem. Soc. 126, 6914–6922 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Wolynes, P. G., Onuchic, J. N. & Thirumalai, D. Navigating the folding routes. Science 267, 1619–1620 (1995)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Onuchic, J. N., Wolynes, P. G., Luthey-Schulten, Z. & Socci, N. D. Toward an outline of the topography of a realistic protein-folding funnel. Proc. Natl Acad. Sci. USA 92, 3626–3630 (1995)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Dill, K. A., Ozkan, S. B., Shell, M. S. & Weikl, T. R. The protein folding problem. Annu. Rev. Biophys. 37, 289–316 (2008)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Nguyen, H. D. & Hall, C. K. Effect of rate of chemical or thermal renaturation on refolding and aggregation of a simple lattice protein. Biotechnol. Bioeng. 80, 823–834 (2002)

    CAS  Article  Google Scholar 

  24. 24

    Ikai, A. & Tanford, C. Kinetic evidence for incorrectly folded intermediate states in the refolding of denatured proteins. Nature 230, 100–102 (1971)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Sela, M., White, F. H., Jr & Anfinsen, C. B. Reductive cleavage of disulfide bridges in ribonuclease. Science 125, 691–692 (1957)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Frauenfelder, H., Sligar, S. G. & Wolynes, P. G. The energy landscapes and motions of proteins. Science 254, 1598–1603 (1991)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Ma, B. & Nussinov, R. Enzyme dynamics point to stepwise conformational selection in catalysis. Curr. Opin. Chem. Biol. 14, 652–659 (2010)

    CAS  Article  Google Scholar 

  28. 28

    Boehr, D. D., Nussinov, R. & Wright, P. E. The role of dynamic conformational ensembles in biomolecular recognition. Nature Chem. Biol. 5, 789–796 (2009)

    CAS  Article  Google Scholar 

  29. 29

    Perkins, T. T., Li, H. W., Dalal, R. V., Gelles, J. & Block, S. M. Forward and reverse motion of single RecBCD molecules on DNA. Biophys. J. 86, 1640–1648 (2004)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Amitani, I., Liu, B., Dombrowski, C. C., Baskin, R. J. & Kowalczykowski, S. C. Watching individual proteins acting on single molecules of DNA. Methods Enzymol. 472, 261–291 (2010)

    CAS  Article  Google Scholar 

  31. 31

    Roman, L. J. & Kowalczykowski, S. C. Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry 28, 2863–2873 (1989)

    CAS  Article  Google Scholar 

  32. 32

    Bianco, P. R. & Kowalczykowski, S. C. The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5′-GCTGGTGG-3′. Proc. Natl Acad. Sci. USA 94, 6706–6711 (1997)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Spies, M., Dillingham, M. S. & Kowalczykowski, S. C. Translocation by the RecB motor is an absolute requirement for χ−recognition and RecA protein loading by RecBCD enzyme. J. Biol. Chem. 280, 37078–37087 (2005)

    CAS  Article  Google Scholar 

  34. 34

    Kreuzer, K. N. & Jongeneel, C. V. Escherichia coli phage T4 topoisomerase. Methods Enzymol. 100, 144–160 (1983)

    CAS  Article  Google Scholar 

  35. 35

    Neuman, K. C. & Block, S. M. Optical trapping. Rev. Sci. Instrum. 75, 2787–2809 (2004)

    ADS  CAS  Article  Google Scholar 

Download references


We are grateful to members of the laboratory for their comments on this work. S.C.K. was supported by the National Institutes of Health (GM-62653 and GM-64745).

Author information




B.L., R.J.B. and S.C.K. conceived the general ideas, designed the experiments and interpreted the data. B.L. performed experiments. B.L. and S.C.K. analysed the data and wrote the manuscript. R.J.B. passed away on July 3, 2010; this work is dedicated to his collegiality and contributions.

Corresponding author

Correspondence to Stephen C. Kowalczykowski.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-10. (PDF 793 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, B., Baskin, R. & Kowalczykowski, S. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature 500, 482–485 (2013). https://doi.org/10.1038/nature12333

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.