The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7.
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.
Hyland
Edel M
EM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Rezende
Lisa F
LF
Richardson
Charles C
CC
D017123:Bacteriophage T7 / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002845:Chromatography; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; D006639:Histidine; D004247:DNA