Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.
Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.
Kim
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YT
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Tabor
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Churchich
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Richardson
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CC
D001483:Base Sequence; D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005454:Fluorescence Polarization; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase