Home LiteratureArticle Details
PMID: 17604719 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase.

Cell ·Vol. 129 ·No. 7 ·2007-06-29 ·Pages 1299-309

Johnson DS, Bai L, Smith BY, Patel SS, Wang MD

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 approximately 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.

MeSH Terms
Bacteriophage T7/genetics Biomechanical Phenomena DNA/biosynthesis,genetics DNA Helicases/genetics DNA Replication/genetics DNA, Single-Stranded/genetics,metabolism Mechanotransduction, Cellular/genetics Models, Molecular Molecular Motor Proteins/genetics Molecular Structure Stress, Mechanical Time Factors
Chemicals
DNA, Single-Stranded Molecular Motor Proteins DNA DNA Helicases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Johnson Daniel S
Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, NY 14853, USA.
Bai Lu
Smith Benjamin Y
Patel Smita S
Wang Michelle D
References (55)
55 references, click to expand
  1. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity.
    Nature. 2003 Jun 19;423(6942):889-93 PMID: 12815437
  2. DNA primase acts as a molecular brake in DNA replication.
    Nature. 2006 Feb 2;439(7076):621-4 PMID: 16452983
  3. A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase.
    Cell. 2003 Sep 5;114(5):647-54 PMID: 13678587
  4. A motor that makes its own track: helicase unwinding of DNA.
    Phys Rev Lett. 2003 Dec 19;91(25):258103 PMID: 14754162
  5. Forward and reverse motion of single RecBCD molecules on DNA.
    Biophys J. 2004 Mar;86(3):1640-8 PMID: 14990491
  6. Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.
    Cell. 1999 Oct 15;99(2):167-77 PMID: 10535735
  7. Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed.
    Biochemistry. 2000 Jan 11;39(1):205-12 PMID: 10625495
  8. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides.
    Cell. 2000 Jun 9;101(6):589-600 PMID: 10892646
  9. Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.
    EMBO J. 2000 Jul 17;19(14):3799-810 PMID: 10899133
  10. Structure and function of hexameric helicases.
    Annu Rev Biochem. 2000;69:651-97 PMID: 10966472
  11. Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules.
    Nature. 2001 Jan 18;409(6818):370-4 PMID: 11201749
  12. Unwinding the 'Gordian knot' of helicase action.
    Trends Biochem Sci. 2001 Jan;26(1):47-54 PMID: 11165517
  13. A general model for nucleic acid helicases and their "coupling" within macromolecular machines.
    Cell. 2001 Jan 26;104(2):177-90 PMID: 11207360
  14. Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork.
    Biochemistry. 2001 Apr 10;40(14):4459-77 PMID: 11284703
  15. Modularity and specialization in superfamily 1 and 2 helicases.
    J Bacteriol. 2002 Apr;184(7):1819-26 PMID: 11889086
  16. Vinylphosphonate internucleotide linkages inhibit the activity of PcrA DNA helicase.
    Biochemistry. 2002 Jun 18;41(24):7725-31 PMID: 12056904
  17. Probing protein-DNA interactions by unzipping a single DNA double helix.
    Biophys J. 2002 Aug;83(2):1098-105 PMID: 12124289
  18. T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA.
    J Mol Biol. 2002 Aug 30;321(5):807-19 PMID: 12206763
  19. Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior.
    Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13538-43 PMID: 12370445
  20. The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity.
    Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):16006-11 PMID: 12441398
  21. A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro.
    J Mol Biol. 2003 Jan 31;325(5):913-35 PMID: 12527299
  22. Laser-induced heating in optical traps.
    Biophys J. 2003 Feb;84(2 Pt 1):1308-16 PMID: 12547811
  23. Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: Structures and properties of isolated helicases.
    Q Rev Biophys. 2002 Nov;35(4):431-78 PMID: 12621862
  24. Single-molecule assay reveals strand switching and enhanced processivity of UvrD.
    Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6439-44 PMID: 15079074
  25. The DNA-unwinding mechanism of the ring helicase of bacteriophage T7.
    Proc Natl Acad Sci U S A. 2004 May 11;101(19):7264-9 PMID: 15123793
  26. Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes.
    Mol Cell. 2004 Aug 13;15(3):453-65 PMID: 15304224
  27. Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.
    Proc Natl Acad Sci U S A. 1981 Jan;78(1):205-9 PMID: 6454135
  28. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule.
    Cell. 1983 Jun;33(2):315-7 PMID: 6344999
  29. DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7.
    J Biol Chem. 1983 Nov 25;258(22):14009-16 PMID: 6139375
  30. The gene 4 protein of bacteriophage T7. Characterization of helicase activity.
    J Biol Chem. 1983 Nov 25;258(22):14017-24 PMID: 6315716
  31. Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding.
    Science. 1992 Apr 17;256(5055):350-5 PMID: 1533057
  32. Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.
    J Biol Chem. 1992 Jul 25;267(21):15013-21 PMID: 1321824
  33. Energy flow considerations and thermal fluctuational opening of DNA base pairs at a replicating fork: unwinding consistent with observed replication rates.
    J Biomol Struct Dyn. 1992 Oct;10(2):415-27 PMID: 1466817
  34. Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins.
    J Biol Chem. 1993 May 15;268(14):10668-75 PMID: 8486715
  35. Escherichia coli rep helicase unwinds DNA by an active mechanism.
    Biochemistry. 1993 Jul 13;32(27):6815-20 PMID: 8392863
  36. A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7754-8 PMID: 7689228
  37. Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.
    Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3869-73 PMID: 7731998
  38. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules.
    Science. 1996 Feb 9;271(5250):795-9 PMID: 8628994
  39. Mechanisms of helicase-catalyzed DNA unwinding.
    Annu Rev Biochem. 1996;65:169-214 PMID: 8811178
  40. A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding.
    Biochemistry. 1997 Nov 18;36(46):14080-7 PMID: 9369480
  41. Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules.
    Nat Struct Mol Biol. 2006 Jun;13(6):549-54 PMID: 16732285
  42. Mechanism of DNA translocation in a replicative hexameric helicase.
    Nature. 2006 Jul 20;442(7100):270-5 PMID: 16855583
  43. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke.
    Cell. 2006 Dec 29;127(7):1349-60 PMID: 17190599
  44. RecBCD enzyme is a bipolar DNA helicase.
    Nature. 2003 Jun 19;423(6942):893-7 PMID: 12815438
  45. Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate.
    J Biol Chem. 1997 Dec 19;272(51):32267-73 PMID: 9405431
  46. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding.
    Structure. 1998 Jan 15;6(1):89-100 PMID: 9493270
  47. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.
    Cell. 1999 Apr 2;97(1):75-84 PMID: 10199404
  48. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.
    Nature. 2004 Nov 11;432(7014):187-93 PMID: 15538360
  49. Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA.
    J Mol Biol. 2004 Dec 10;344(5):1287-309 PMID: 15561144
  50. Opening of nucleic-acid double strands by helicases: active versus passive opening.
    Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Jan;71(1 Pt 1):011904 PMID: 15697627
  51. DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase.
    Nature. 2005 May 19;435(7040):370-3 PMID: 15902262
  52. DINAMelt web server for nucleic acid melting prediction.
    Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W577-81 PMID: 15980540
  53. Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain.
    Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10076-81 PMID: 16009938
  54. Repetitive shuttling of a motor protein on DNA.
    Nature. 2005 Oct 27;437(7063):1321-5 PMID: 16251956
  55. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP.
    Nature. 2006 Jan 5;439(7072):105-8 PMID: 16397502
Article Info
Journal
Cell
Abbr.
Cell
ISSN
0092-8674
DOI
10.1016/j.cell.2007.04.038
Published
2007-06-29
Pages
1299-309
Language
English
Region
United States
NLM ID
0413066
PMCID
PMC2699903
Subset
IM
Grants
NIGMS NIH HHS · R01 GM059849-05 · United States
NIGMS NIH HHS · R01 GM059849 · United States
NIGMS NIH HHS · R01 GM059849-04 · United States
NIGMS NIH HHS · R01 GM055310 · United States
NIGMS NIH HHS · R01 GM059849-07 · United States
NIGMS NIH HHS · R01 GM059849-09 · United States
NIGMS NIH HHS · GM55310 · United States
NIGMS NIH HHS · R01 GM059849-06A1 · United States
NIGMS NIH HHS · R01 GM059849-08 · United States
Corrections
CommentIn
PMID: 17604712
External Links
PubMed source DOI: 10.1016/j.cell.2007.04.038 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2