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Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor

Nature Structural & Molecular Biology volume 17, pages 1210–1217 (2010)Cite this article

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Abstract

E. coli RecBCD is a DNA helicase with two ATPase motors (RecB, a 3′→5′ translocase, and RecD, a 5′→3′ translocase) that function in repair of double-stranded DNA breaks. The RecBC heterodimer, with only the RecB motor, remains a processive helicase. Here we examined RecBC translocation along single-stranded DNA (ssDNA). Notably, we found RecBC to have two translocase activities: the primary translocase moves 3′→5′, whereas the secondary translocase moves RecBC along the opposite strand of a forked DNA at a similar rate. The secondary translocase is insensitive to the ssDNA backbone polarity, and we propose that it may fuel RecBCD translocation along double-stranded DNA ahead of the unwinding fork and ensure that the unwound single strands move through RecBCD at the same rate after interaction with a crossover hot-spot indicator (Chi) sequence.

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Figure 1: RecBCD and RecBC structures.
Figure 2: RecB translocates with 3′→5′ directionality along ssDNA.
Figure 3: RecBC has both primary (3′→5′) and secondary (5′→3′) translocase activities.
Figure 4: The primary RecBC translocase site remains bound to ssDNA upon reaching a 5′ end, while its secondary translocase continues.
Figure 5: Primary and secondary RecBC translocases operate simultaneously along two ssDNA extensions.
Figure 6: RecBC re-initiation of DNA unwinding after a ssDNA gap.

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References

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

    Article  CAS  Google Scholar 

  2. Singleton, M.R., Dillingham, M.S. & Wigley, D.B. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Rigden, D.J. An inactivated nuclease-like domain in RecC with novel function: implications for evolution. BMC Struct. Biol. 5, 9 (2005).

    Article  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Bianco, P.R. & Kowalczykowski, S.C. Translocation step size and mechanism of the RecBC DNA helicase. Nature 405, 368–372 (2000).

    Article  CAS  Google Scholar 

  9. Korangy, F. & Julin, D.A. Efficiency of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry 33, 9552–9560 (1994).

    Article  CAS  Google Scholar 

  10. Wu, C.G. & Lohman, T.M. Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases. J. Mol. Biol. 382, 312–326 (2008).

    Article  CAS  Google Scholar 

  11. Ganesan, S. & Smith, G.R. Strand-specific binding to duplex DNA ends by the subunits of the Escherichia coli RecBCD enzyme. J. Mol. Biol. 229, 67–78 (1993).

    Article  CAS  Google Scholar 

  12. Farah, J.A. & Smith, G.R. The RecBCD enzyme initiation complex for DNA unwinding: enzyme positioning and DNA opening. J. Mol. Biol. 272, 699–715 (1997).

    Article  CAS  Google Scholar 

  13. 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 

  14. 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 

  15. Taylor, A. & Smith, G.R. Unwinding and rewinding of DNA by the RecBC enzyme. Cell 22, 447–457 (1980).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Lucius, A.L. et al. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies. J. Mol. Biol. 324, 409–428 (2002).

    Article  CAS  Google Scholar 

  19. Lucius, A.L. & Lohman, T.M. Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E. coli RecBCD helicase-catalyzed DNA unwinding. J. Mol. Biol. 339, 751–771 (2004).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Saikrishnan, K., Griffiths, S.P., Cook, N., Court, R. & Wigley, D.B. DNA binding to RecD: role of the 1B domain in SF1B helicase activity. EMBO J. 27, 2222–2229 (2008).

    Article  CAS  Google Scholar 

  22. Wong, C.J. & Lohman, T.M. Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC. J. Mol. Biol. 378, 761–777 (2008).

    Article  Google Scholar 

  23. Boehmer, P.E. & Emmerson, P.T. The RecB subunit of the Escherichia coli RecBCD enzyme couples ATP hydrolysis to DNA unwinding. J. Biol. Chem. 267, 4981–4987 (1992).

    CAS  PubMed  Google Scholar 

  24. Phillips, R.J., Hickleton, D.C., Boehmer, P.E. & Emmerson, P.T. The RecB protein of Escherichia coli translocates along single-stranded DNA in the 3′ to 5′ direction: a proposed ratchet mechanism. Mol. Gen. Genet. 254, 319–329 (1997).

    CAS  PubMed  Google Scholar 

  25. Fischer, C.J., Maluf, N.K. & Lohman, T.M. Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA. J. Mol. Biol. 344, 1287–1309 (2004).

    Article  CAS  Google Scholar 

  26. Fischer, C.J. & Lohman, T.M. ATP-dependent translocation of proteins along single-stranded DNA: models and methods of analysis of pre-steady state kinetics. J. Mol. Biol. 344, 1265–1286 (2004).

    Article  CAS  Google Scholar 

  27. Lucius, A.L., Maluf, N.K., Fischer, C.J. & Lohman, T.M. General methods for analysis of sequential “n-step” kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding. Biophys. J. 85, 2224–2239 (2003).

    Article  CAS  Google Scholar 

  28. Hickson, I.D., Robson, C.N., Atkinson, K.E., Hutton, L. & Emmerson, P.T. Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins. J. Biol. Chem. 260, 1224–1229 (1985).

    CAS  PubMed  Google Scholar 

  29. Brendza, K.M. et al. Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain. Proc. Natl. Acad. Sci. USA 102, 10076–10081 (2005).

    Article  CAS  Google Scholar 

  30. Dillingham, M.S., Wigley, D.B. & Webb, M.R. Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine. Biochemistry 41, 643–651 (2002).

    Article  CAS  Google Scholar 

  31. Niedziela-Majka, A., Chesnik, M.A., Tomko, E.J. & Lohman, T.M. Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro. J. Biol. Chem. 282, 27076–27085 (2007).

    Article  CAS  Google Scholar 

  32. Tomko, E.J., Fischer, C.J., Niedziela-Majka, A. & Lohman, T.M. A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA. Mol. Cell 26, 335–347 (2007).

    Article  CAS  Google Scholar 

  33. Antony, E. et al. Srs2 disassembles Rad51 filaments by a protein-protein interaction triggering ATP turnover and dissociation of Rad51 from DNA. Mol. Cell 35, 105–115 (2009).

    Article  CAS  Google Scholar 

  34. Yeeles, J.T. & Dillingham, M.S.A. Dual-nuclease mechanism for DNA break processing by AddAB-type helicase-nucleases. J. Mol. Biol. 371, 66–78 (2007).

    Article  CAS  Google Scholar 

  35. Unciuleac, M.C. & Shuman, S. Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines. J. Biol. Chem. 285, 2632–2641 (2010).

    Article  CAS  Google Scholar 

  36. Lucius, A.L., Jason Wong, C. & 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 

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Acknowledgements

We thank R. Galletto, C. Fischer, A. Lucius, K. Maluf, E. Galburt, N. Baker, P. Burgers, G. Smith, T. Ellenberger, S. Kowalczykowski and E. Antony for valuable discussions and comments on the manuscript, G. Smith (Fred Hutchinson Cancer Research Center), A. Taylor (Fred Hutchinson Cancer Research Center) and D. Julin (University of Maryland, College Park) for plasmids and cell lines and T. Ho (Washington University School of Medicine) for synthesis and purification of DNA. This work was supported in part by US National Institutes of Health grant GM045948 (to T.M.L.).

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  1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri, USA

    Colin G Wu, Christina Bradford & Timothy M Lohman

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  1. Colin G Wu
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  2. Christina Bradford
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Contributions

C.G.W. and T.M.L. designed the experiments. C.G.W. purified the protein, carried out the stopped-flow and DNA-unwinding experiments and analyzed the data; C.B. and C.G.W. carried out the fluorescence resonance energy transfer and RecBC translocation experiments as a function of NaCl concentration. T.M.L. supervised the study, and C.G.W. and T.M.L. wrote the manuscript.

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Correspondence to Timothy M Lohman.

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The authors declare no competing financial interests.

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Supplementary Methods, Supplementary Figures 1–6, Supplementary Tables 1–3 and Supplementary Schemes 1–3 (including Supplementary Equations 1–3) (PDF 750 kb)

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Wu, C., Bradford, C. & Lohman, T. Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor. Nat Struct Mol Biol 17, 1210–1217 (2010). https://doi.org/10.1038/nsmb.1901

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