Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template

Cell. 1991 Apr 19;65(2):249-58. doi: 10.1016/0092-8674(91)90159-v.


T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Base Sequence
  • Cross-Linking Reagents
  • DNA / chemical synthesis
  • DNA / metabolism
  • DNA Helicases / metabolism*
  • DNA Replication
  • DNA, Viral / biosynthesis
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism*
  • Models, Structural
  • Molecular Sequence Data
  • T-Phages / enzymology*
  • T-Phages / genetics
  • Templates, Genetic


  • Cross-Linking Reagents
  • DNA, Viral
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Triphosphate
  • DNA
  • DNA-Directed DNA Polymerase
  • DNA Helicases