Mechanism of bacteriophage T4 DNA holoenzyme assembly: the 44/62 protein acts as a molecular motor

Biochemistry. 1997 Mar 11;36(10):2733-43. doi: 10.1021/bi962139l.


The role of ATP hydrolysis by the 44/62 protein in formation of the stable holoenzyme DNA replication complex has been further elucidated by specifically examining the role that the 44/62 protein plays in loading the 45 protein onto the DNA substrate. A stable phospho-45 protein or phosphorylated holoenzyme complex was not detected or isolated, suggesting that the 44/62 protein may not act as a protein kinase. Product and dead-end inhibition data are consistent with an ordered kinetic mechanism with respect to product release in which phosphate is released from the 44/62 protein prior to ADP. Positional isotope effect studies support this mechanism and failed to demonstrate that ATP hydrolysis by the 44/62 protein is reversible. Steady-state ATPase assays using aluminum tetrafluoride as an inhibitor are also consistent with release of ADP being partially rate-limiting. Aluminum tetrafluoride acts to trap ADP on the enzyme after turnover, forming a stable transition state analog that dissociates slowly from the enzyme. Processive DNA synthesis does not occur using the accessory proteins in the presence of pre- or post-hydrolysis analogs of ATP nor in the presence of ADP-AlF4, indicating that turnover of the 44/62 protein is absolutely required for formation of the holoenzyme complex. Collectively, data obtained regarding ATP hydrolysis by the 44/62 protein are described in terms of the clamp loading protein functioning as a molecular motor, similar to other systems including myosin and kinesin.

Publication types

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

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Aluminum Compounds / pharmacology
  • Bacteriophage T4 / enzymology*
  • Chromatography, Gel
  • Coenzymes / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology
  • Fluorides / pharmacology
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Phosphates / metabolism
  • Phosphorylation
  • Protein Conformation
  • Trans-Activators / metabolism
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism*


  • Aluminum Compounds
  • Coenzymes
  • Enzyme Inhibitors
  • Phosphates
  • Trans-Activators
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • gene 44 protein, Enterobacteria phage T4
  • gene 45 protein, Enterobacteria phage T4
  • gp62 protein, bacteriophage T4
  • tetrafluoroaluminate
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • DNA-Directed DNA Polymerase
  • Fluorides