Direct observation of translocation in individual DNA polymerase complexes

J Biol Chem. 2012 Apr 13;287(16):13407-21. doi: 10.1074/jbc.M111.338418. Epub 2012 Feb 29.


Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / metabolism
  • Bacillus Phages / enzymology*
  • Bacillus Phages / genetics*
  • Catalytic Domain / physiology
  • DNA Replication / physiology*
  • DNA, Viral / metabolism
  • DNA-Directed DNA Polymerase / chemical synthesis
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism*
  • Diphosphates / metabolism
  • Enzyme Activation / physiology
  • Exonucleases / metabolism
  • Hemolysin Proteins / chemistry
  • Hemolysin Proteins / metabolism
  • Inverted Repeat Sequences / genetics
  • Molecular Motor Proteins / physiology
  • Nanopores*
  • Nucleic Acid Conformation


  • ATP-Binding Cassette Transporters
  • DNA, Viral
  • Diphosphates
  • Hemolysin Proteins
  • Molecular Motor Proteins
  • diphosphoric acid
  • DNA-Directed DNA Polymerase
  • Exonucleases
  • hemolysin B