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Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Journal Article


Abstract


  • The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

Authors


  •   Etson, Candice M. (external author)
  •   Hamdan, Samir M. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2010

Citation


  • Etson, C. M., Hamdan, S. M., Richardson, C. C. & van Oijen, A. M. (2010). Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. Proceedings of the National Academy of Sciences of USA, 107 (5), 1900-1905.

Scopus Eid


  • 2-s2.0-76649145153

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2179

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 1900

End Page


  • 1905

Volume


  • 107

Issue


  • 5

Place Of Publication


  • United States

Abstract


  • The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

Authors


  •   Etson, Candice M. (external author)
  •   Hamdan, Samir M. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2010

Citation


  • Etson, C. M., Hamdan, S. M., Richardson, C. C. & van Oijen, A. M. (2010). Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. Proceedings of the National Academy of Sciences of USA, 107 (5), 1900-1905.

Scopus Eid


  • 2-s2.0-76649145153

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2179

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 1900

End Page


  • 1905

Volume


  • 107

Issue


  • 5

Place Of Publication


  • United States