Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

Daniel S. Johnson; Lu Bai; Benjamin Y. Smith; Smita S. Patel; Michelle D. Wang

doi:10.1016/j.cell.2007.04.038

Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

Daniel S. Johnson, Lu Bai, Benjamin Y. Smith, Smita S. Patel, Michelle D. Wang

Biochemistry & Molecular Biology

Research output: Contribution to journal › Article › peer-review

206 Scopus citations

Abstract

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased ∼10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.

Original language	English (US)
Pages (from-to)	1299-1309
Number of pages	11
Journal	Cell
Volume	129
Issue number	7
DOIs	https://doi.org/10.1016/j.cell.2007.04.038
State	Published - Jun 29 2007

All Science Journal Classification (ASJC) codes

General Biochemistry, Genetics and Molecular Biology

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title = "Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase",

abstract = "Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased ∼10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.",

author = "Johnson, {Daniel S.} and Lu Bai and Smith, {Benjamin Y.} and Patel, {Smita S.} and Wang, {Michelle D.}",

note = "Funding Information: We thank members of the Wang lab for helpful discussions and critical reading of the manuscript. We also thank R.C. Yeh, S. J. Koch, D.A. Wacker, and A. Shundrovsky for assistance during the initial stage of the project. We thank Y.J. Jeong and I. Donmez for providing purified T7 helicase protein. This research was supported by the Keck Foundation to M.D.W., GM55310 NIH grant to S.S.P., and a Molecular Biophysics Training Grant to Cornell. ",

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AU - Johnson, Daniel S.

AU - Bai, Lu

AU - Smith, Benjamin Y.

AU - Patel, Smita S.

AU - Wang, Michelle D.

N1 - Funding Information: We thank members of the Wang lab for helpful discussions and critical reading of the manuscript. We also thank R.C. Yeh, S. J. Koch, D.A. Wacker, and A. Shundrovsky for assistance during the initial stage of the project. We thank Y.J. Jeong and I. Donmez for providing purified T7 helicase protein. This research was supported by the Keck Foundation to M.D.W., GM55310 NIH grant to S.S.P., and a Molecular Biophysics Training Grant to Cornell.

PY - 2007/6/29

Y1 - 2007/6/29

N2 - Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased ∼10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.

AB - Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased ∼10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.

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Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

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