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Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Author

Listed:
  • Kyung Suk Lee

    (Center for Physics in Living Cells and Institute for Genomic Biology, University of Illinois, Urbana-Champaign)

  • Hamza Balci

    (Kent State University)

  • Haifeng Jia

    (Washington University School of Medicine)

  • Timothy M. Lohman

    (Washington University School of Medicine)

  • Taekjip Ha

    (Center for Physics in Living Cells and Institute for Genomic Biology, University of Illinois, Urbana-Champaign
    Howard Hughes Medical Institute, University of Illinois)

Abstract

Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding.

Suggested Citation

  • Kyung Suk Lee & Hamza Balci & Haifeng Jia & Timothy M. Lohman & Taekjip Ha, 2013. "Direct imaging of single UvrD helicase dynamics on long single-stranded DNA," Nature Communications, Nature, vol. 4(1), pages 1-9, October.
    • Handle: RePEc:nat:natcom:v:4:y:2013:i:1:d:10.1038_ncomms2882
    DOI: 10.1038/ncomms2882
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    Cited by:

    1. Sean P. Carney & Wen Ma & Kevin D. Whitley & Haifeng Jia & Timothy M. Lohman & Zaida Luthey-Schulten & Yann R. Chemla, 2021. "Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
    2. Xiao-Wen Yang & Xiao-Peng Han & Chong Han & James London & Richard Fishel & Jiaquan Liu, 2022. "MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair," Nature Communications, Nature, vol. 13(1), pages 1-17, December.

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