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Kim, YT, Tabor, S, Bortner, C, Griffith, JD and Richardson, CC (1992) Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein. J. Biol. Chem. 267:15022-31


Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene. Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562. Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative. Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA. The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA. In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively. Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25. Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.




Adenosine Triphosphate/metabolism; Amino Acid Sequence; Binding Sites; Cell Line; Chromatography, DEAE-Cellulose; Chromatography, Gel; DNA, Single-Stranded/metabolism; DNA-Binding Proteins/isolation & purification; DNA-Binding Proteins/metabolism; DNA-Binding Proteins/ultrastructure; Electrophoresis, Polyacrylamide Gel; Genes, Viral; Isoelectric Focusing; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; Plasmids; Sequence Alignment; Spectrometry, Fluorescence; T-Phages/genetics; T-Phages/metabolism; Viral Proteins/isolation & purification; Viral Proteins/metabolism; Viral Proteins/ultrastructure



Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status


GO:0003674: molecular function



Figure 3: A nitrocellulose filter was used to determine levels of binding between gp2.5 and radioactively labeled DNA. Nitrocellulose is negatively charged, so only DNA bound to gp2.5 could be retained. With increasing concentrations of gp2.5, greater amounts of single-stranded DNA were retained. The experiment only demonstrated a small amount of binding between gp2.5 and double stranded DNA. This suggests that gp2.5's molecular function is the binding of ssDNA.

CACAO 12008


See also


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