The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase

J Biol Chem. 2003 Aug 8;278(32):29538-45. doi: 10.1074/jbc.M304318200. Epub 2003 May 24.


Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.

Publication types

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

MeSH terms

  • Bacteriophage T7 / chemistry*
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA / metabolism
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / physiology*
  • DNA-Directed DNA Polymerase / chemistry*
  • Dimerization
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Microscopy, Electron
  • Mutagenesis, Site-Directed
  • Mutation
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Surface Plasmon Resonance
  • Time Factors
  • Viral Proteins / chemistry*
  • Viral Proteins / physiology*


  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • gene 2.5 protein, Enterobacteria phage T7
  • DNA
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase