Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

Nature Chemical Biology volume 7, pages 367–374 (2011)Cite this article

Subjects

Abstract

λ exonuclease degrades one strand of duplex DNA in the 5′-to-3′ direction to generate a 3′ overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3′ overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick–paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5′ end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Single-molecule FRET assay for λ exonuclease activity.
Figure 2: λ exonuclease carries out initiation and distributive degradation until it is stably engaged by the substrate.
Figure 3: After stable engagement with the 3′ overhang, λ exonuclease processively degrades the substrate without dissociating.
Figure 4: λ exonuclease performs distributive degradation before complete engagement to DNA.
Figure 5: DNA mismatches impede the degradation activity of λ exonuclease in a synergistic manner.
Figure 6: Degradation arrest on a bubble DNA and escape from a known pausing sequence.

Similar content being viewed by others

References

  1. Ceska, T.A. & Sayers, J.R. Structure-specific DNA cleavage by 5′ nucleases. Trends Biochem. Sci. 23, 331–336 (1998).

    Article  CAS  Google Scholar 

  2. Haber, J.E. In-vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases. Bioessays 17, 609–620 (1995).

    Article  CAS  Google Scholar 

  3. Little, J.W., Lehman, I.R. & Kaiser, A.D. An exonuclease induced by bacteriophage λ. I. Preparation of crystalline enzyme. J. Biol. Chem. 242, 672–678 (1967).

    CAS  PubMed  Google Scholar 

  4. Little, J.W. An exonuclease induced by bacteriophage λ. II. Nature of enzymatic reaction. J. Biol. Chem. 242, 679–686 (1967).

    CAS  PubMed  Google Scholar 

  5. Carter, D.M. & Radding, C.M. Role of exonuclease and β protein of phage λ in genetic recombination. II. Substrate specificity and mode of action of λ exonuclease. J. Biol. Chem. 246, 2502–2512 (1971).

    CAS  PubMed  Google Scholar 

  6. Thomas, K.R. & Olivera, B.M. Processivity of DNA exo-nucleases. J. Biol. Chem. 253, 424–429 (1978).

    CAS  PubMed  Google Scholar 

  7. Radding, C.M. Regulation of lambda exonuclease. I. Properties of lambda exonuclease purified from lysogens of lambda T11 and wild type. J. Mol. Biol. 18, 235–250 (1966).

    Article  CAS  Google Scholar 

  8. Black, L.W. DNA packaging in dsDNA bacteriophages. Annu. Rev. Microbiol. 43, 267–292 (1989).

    Article  CAS  Google Scholar 

  9. Muyrers, J.P.P., Zhang, Y.M., Buchholz, F. & Stewart, A.F. RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev. 14, 1971–1982 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hingorani, M.M. & O'Donnell, M. Toroidal proteins: running rings around DNA. Curr. Biol. 8, R83–R86 (1998).

    Article  CAS  Google Scholar 

  11. Guissani, A. Processive and synchronous mechanism of polynucleotide phosphorylase phosphorolysis: a comparison between experimental results and those calculated from a theoretical-study. Eur. J. Biochem. 79, 233–243 (1977).

    Article  CAS  Google Scholar 

  12. Klee, C.B. & Singer, M.F. Processive degradation of individual polyribonucleotide chains. II. Micrococcus lysodeikticus polynucleotide phosphorylase. J. Biol. Chem. 243, 923–927 (1968).

    CAS  PubMed  Google Scholar 

  13. Subramanian, K., Rutvisuttinunt, W., Scott, W. & Myers, R.S. The enzymatic basis of processivity in λ exonuclease. Nucleic Acids Res. 31, 1585–1596 (2003).

    Article  CAS  Google Scholar 

  14. Sriprakash, K.S., Lundh, N., Mooonhuh, M. & Radding, C.M. Specificity of λ exonuclease: interactions with single-stranded DNA. J. Biol. Chem. 250, 5438–5445 (1975).

    CAS  PubMed  Google Scholar 

  15. Mitsis, P.G. & Kwagh, J.G. Characterization of the interaction of lambda exonuclease with the ends of DNA. Nucleic Acids Res. 27, 3057–3063 (1999).

    Article  CAS  Google Scholar 

  16. Dapprich, J. Single-molecule DNA digestion by lambda-exonuclease. Cytometry 36, 163–168 (1999).

    Article  CAS  Google Scholar 

  17. Perkins, T.T., Dalal, R.V., Mitsis, P.G. & Block, S.M. Sequence-dependent pausing of single lambda exonuclease molecules. Science 301, 1914–1918 (2003).

    Article  CAS  Google Scholar 

  18. van Oijen, A.M. et al. Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science 301, 1235–1238 (2003).

    Article  CAS  Google Scholar 

  19. Young, B.A., Gruber, T.M. & Gross, C.A. Views of transcription initiation. Cell 109, 417–420 (2002).

    Article  CAS  Google Scholar 

  20. Marshall, R.A., Aitken, C.E., Dorywalska, M. & Puglisi, J.D. Translation at the single-molecule level. Annu. Rev. Biochem. 77, 177–203 (2008).

    Article  CAS  Google Scholar 

  21. Lucius, A.L., Wong, C.J. & Lohman, T.M. Fluorescence stopped-flow studies of single turnover kinetics of E. coli RecBCD helicase-catalyzed DNA unwinding. J. Mol. Biol. 339, 731–750 (2004).

    Article  CAS  Google Scholar 

  22. Wong, C.J., Lucius, A.L. & Lohman, T.M. Energetics of DNA end binding by E. coli RecBC and RecBCD helicases indicate loop formation in the 3′-single-stranded DNA tail. J. Mol. Biol. 352, 765–782 (2005).

    Article  CAS  Google Scholar 

  23. Wong, C.J., Rice, R.L., Baker, N.A., Ju, T. & Lohman, T.M. Probing 3′-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for “Chi” recognition complexes. J. Mol. Biol. 362, 26–43 (2006).

    Article  CAS  Google Scholar 

  24. Zhuang, X. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000).

    Article  CAS  Google Scholar 

  25. Zhuang, X. et al. Correlating structural dynamics and function in single ribozyme molecules. Science 296, 1473–1476 (2002).

    Article  CAS  Google Scholar 

  26. Rothwell, P.J. et al. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase: primer/template complexes. Proc. Natl. Acad. Sci. USA 100, 1655–1660 (2003).

    Article  CAS  Google Scholar 

  27. Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318, 279–283 (2007).

    Article  CAS  Google Scholar 

  28. Cecconi, C., Shank, E.A., Bustamante, C. & Marqusee, S. Direct observation of the three-state folding of a single protein molecule. Science 309, 2057–2060 (2005).

    Article  CAS  Google Scholar 

  29. Woodside, M.T. et al. Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314, 1001–1004 (2006).

    Article  CAS  Google Scholar 

  30. Shi, J., Dertouzos, J., Gafni, A., Steel, D. & Palfey, B.A. Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis. Proc. Natl. Acad. Sci. USA 103, 5775–5780 (2006).

    Article  CAS  Google Scholar 

  31. Stryer, L. & Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

    Article  CAS  Google Scholar 

  32. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. USA 93, 6264–6268 (1996).

    Article  CAS  Google Scholar 

  33. Kovall, R. & Matthews, B.W. Toroidal structure of lambda-exonuclease. Science 277, 1824–1827 (1997).

    Article  CAS  Google Scholar 

  34. Shaevitz, J.W., Abbondanzieri, E.A., Landick, R. & Block, S.M. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003).

    Article  CAS  Google Scholar 

  35. Murphy, M.C., Rasnik, I., Cheng, W., Lohman, T.M. & Ha, T.J. Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004).

    Article  CAS  Google Scholar 

  36. Selvin, P. & Ha, T. Single-Molecule Techniques: A Laboratory Manual 1st edn. (eds. Selvin, P.R. & Ha, T.) 3–36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2008).

  37. Maluf, N.K., Fischer, C.J. & Lohman, T.M. A dimer of Escherichia coli UvrD is the active form of the helicase in vitro. J. Mol. Biol. 325, 913–935 (2003).

    Article  CAS  Google Scholar 

  38. Luo, G., Wang, M., Konigsberg, W.H. & Xie, X.S. Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 104, 12610–12615 (2007).

    Article  CAS  Google Scholar 

  39. Myong, S. et al. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA. Science 323, 1070–1074 (2009).

    Article  CAS  Google Scholar 

  40. Yin, J. et al. Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling. Chem. Biol. 12, 999–1006 (2005).

    Article  CAS  Google Scholar 

  41. Yin, J., Lin, A.J., Golan, D.E. & Walsh, C.T. Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat. Protoc. 1, 280–285 (2006).

    Article  CAS  Google Scholar 

  42. Hsu, L.M. Promoter clearance and escape in prokaryotes. Biochim. Biophys. Acta 1577, 191–207 (2002).

    Article  CAS  Google Scholar 

  43. Roberts, J.W. RNA polymerase, a scrunching machine. Science 314, 1097–1098 (2006).

    Article  CAS  Google Scholar 

  44. Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002).

    Article  CAS  Google Scholar 

  45. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  46. Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and longlasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    Article  CAS  Google Scholar 

  47. Grossman, D. & van Hoof, A. RNase II structure completes group portrait of 3′ exoribonucleases. Nat. Struct. Mol. Biol. 13, 760–761 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Roy and X. Shi for experimental help, and J. Park for helpful discussions. G.L. was supported by the Jane Coffin Childs Medical Institute. Funds were provided by grants from the US National Science Foundation (0646550, 0822613) and the US National Institutes of Health (GM065367). T.H. is an investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Physics and the Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Gwangrog Lee, Jungmin Yoo, Benjamin J Leslie & Taekjip Ha

  2. Howard Hughes Medical Institute, Urbana, 61801, Illinois, USA

    Gwangrog Lee, Benjamin J Leslie & Taekjip Ha

Authors
  1. Gwangrog Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jungmin Yoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benjamin J Leslie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taekjip Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.L. performed single-molecule and ensemble fluorescent experiments. J.Y. performed single-molecule experiments. G.L. and B.J.L. expressed and purified λ exonuclease, and B.J.L. carried out the protein labeling. G.L., J.Y., B.J.L. and T.H. designed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Taekjip Ha.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figs. 1–14, Supplementary Table 1 (PDF 887 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, G., Yoo, J., Leslie, B. et al. Single-molecule analysis reveals three phases of DNA degradation by an exonuclease. Nat Chem Biol 7, 367–374 (2011). https://doi.org/10.1038/nchembio.561

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.561

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing