Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA.
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, 11 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.
Etson
Candice M
CM
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Hamdan
Samir M
SM
Richardson
Charles C
CC
van Oijen
Antoine M
AM
D017123:Bacteriophage T7 / Q000201:enzymology; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D055459:Facilitated Diffusion; D005456:Fluorescent Dyes; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D055672:Static Electricity; D013879:Thioredoxins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D013879:Thioredoxins; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase