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ATP-induced helicase slippage reveals highly coordinated subunits

Nature volume 478, pages 132–135 (2011)Cite this article

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Abstract

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination1,2. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5′ to 3′ along single-stranded (ss)DNA2,3,4,5,6. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate1,7. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates Vmax, Michaelis constants KM and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

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Figure 1: Comparison of helicase unwinding behaviours with different nucleotides.
Figure 2: Helicase unwinding kinetics.
Figure 3: Processivity dependence on nucleotides and a proposed coordinated model.

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  • 06 October 2011

    The labelling of Fig. 1d and Fig. 3a was corrected.

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Acknowledgements

We thank members of the Wang laboratory for critical reading of the manuscript. We also thank M. A. Hall for assistance with single-molecule assays, data acquisition and data analysis. We wish to acknowledge support from National Institutes of Health grants (GM059849 to M.D.W.; GM55310 to S.S.P.), National Science Foundation grant (MCB-0820293 to M.D.W.) and Cornell’s Molecular Biophysics Training Grant (T32GM008267) Traineeship (to D.S.J. and B.Y.S.).

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Author notes

  1. Daniel S. Johnson

    Present address: Present address: Rockefeller University, New York, New York 10065, USA.,

  2. Bo Sun and Daniel S. Johnson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics - Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, 14853, New York, USA

    Bo Sun, Daniel S. Johnson, Benjamin Y. Smith & Michelle D. Wang

  2. Howard Hughes Medical Institute, Cornell University, Ithaca, 14853, New York, USA

    Bo Sun, Daniel S. Johnson, Benjamin Y. Smith & Michelle D. Wang

  3. Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway, 08854, New Jersey, USA

    Gayatri Patel, Manjula Pandey & Smita S. Patel

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Contributions

B.S., D.S.J., S.S.P. and M.D.W. designed the experiments. D.S.J. found the helicase slippage with ATP. B.S. carried out all single-molecule work and, together with B.Y.S., analysed and interpreted single-molecule data. G.P. performed all the ensemble experiments. M.P. and G.P. purified and analysed the wild-type and mutant T7 gp4 proteins. M.D.W. formulated the theoretical models. B.S., D.S.J., B.Y.S., S.S.P. and M.D.W. wrote the manuscript.

Corresponding authors

Correspondence to Smita S. Patel or Michelle D. Wang.

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The authors declare no competing financial interests.

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The file contains Supplementary Figures 1-13 with legends, a Supplementary Discussion and additional references. (PDF 1587 kb)

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Sun, B., Johnson, D., Patel, G. et al. ATP-induced helicase slippage reveals highly coordinated subunits. Nature 478, 132–135 (2011). https://doi.org/10.1038/nature10409

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