Function and assembly of the bacteriophage T4 DNA replication complex: interactions of the T4 polymerase with various model DNA constructs

J Biol Chem. 2003 Jul 11;278(28):25435-47. doi: 10.1074/jbc.M303370200. Epub 2003 Apr 16.


Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5' --> 3' DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single- and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (Kd approximately 150 nm) and a binding site size of approximately 10 nucleotides for single-stranded DNA and approximately 13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (Kd approximately 5 nm) to DNA constructs carrying a primer-template junction, with the polymerase covering approximately 5 nucleotides downstream and approximately 6-7 bp upstream of the 3'-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.

Publication types

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

MeSH terms

  • Anisotropy
  • Antigens, Neoplasm / chemistry*
  • Antigens, Neoplasm / metabolism
  • Bacteriophage T4 / genetics*
  • Bacteriophage T4 / physiology*
  • Binding Sites
  • DNA / metabolism*
  • DNA Replication*
  • DNA, Single-Stranded / metabolism
  • Kinetics
  • Oligonucleotides / chemistry
  • Protein Binding
  • Software
  • Spectrometry, Fluorescence
  • Surface Plasmon Resonance
  • Thermodynamics
  • Time Factors


  • Antigens, Neoplasm
  • DNA, Single-Stranded
  • Oligonucleotides
  • tumor-associated antigen, gp43
  • DNA