Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Candice M. Etson, Samir Hamdan, Charles C. Richardson, Antoine M. Van Oijen

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1900-1905
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number5
DOIs
StatePublished - Feb 2 2010

Keywords

  • DNA replication
  • Facilitated diffusion
  • Processivity
  • Single-molecule imaging
  • Sliding

ASJC Scopus subject areas

  • General

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