The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7

J Biol Chem. 2003 Feb 28;278(9):7247-56. doi: 10.1074/jbc.M210605200. Epub 2002 Dec 20.

Abstract

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.

Publication types

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

MeSH terms

  • Bacteriophage T7 / chemistry*
  • Bacteriophage T7 / metabolism
  • Binding Sites
  • Chromatography
  • Chromatography, Gel
  • Crystallography, X-Ray
  • DNA / metabolism*
  • DNA, Single-Stranded
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics*
  • Dimerization
  • Escherichia coli / metabolism
  • Histidine / chemistry
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Oligonucleotides / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Surface Plasmon Resonance
  • Time Factors
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics*

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Oligonucleotides
  • Recombinant Fusion Proteins
  • Viral Proteins
  • gene 2.5 protein, Enterobacteria phage T7
  • Histidine
  • DNA