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

Edel M Hyland, Lisa F Rezende, Charles C Richardson

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)7247-56
Number of pages10
JournalThe Journal of biological chemistry
Volume278
Issue number9
DOIs
Publication statusPublished - 28 Feb 2003
Externally publishedYes

Keywords

  • Bacteriophage T7/chemistry
  • Binding Sites
  • Chromatography
  • Chromatography, Gel
  • Crystallography, X-Ray
  • DNA/metabolism
  • DNA, Single-Stranded
  • DNA-Binding Proteins/chemistry
  • 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

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