DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction

J Biol Chem. 1991 Oct 25;266(30):20034-44.


We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Autoradiography
  • Base Sequence
  • DNA Fingerprinting
  • DNA Polymerase III
  • DNA, Viral / genetics
  • DNA-Directed DNA Polymerase / genetics*
  • Electrophoresis, Polyacrylamide Gel
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • T-Phages / enzymology*
  • Templates, Genetic*


  • DNA, Viral
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase