Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69

J Biol Chem. 2003 Feb 7;278(6):3876-81. doi: 10.1074/jbc.M210497200. Epub 2002 Nov 27.


The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophages / genetics
  • Bacteriophages / metabolism*
  • Cloning, Molecular
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Protein Binding
  • Spectrometry, Fluorescence


  • DNA-Binding Proteins
  • DNA-Directed DNA Polymerase