Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.
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.
Kim
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YT
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Tabor
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Bortner
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Griffith
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JD
Richardson
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D007525:Isoelectric Focusing; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate