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Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Authors
  • Kim, Y T
  • Tabor, S
  • Churchich, J E
  • Richardson, C C
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry & Molecular Biology (ASBMB)
Publication Date
Jul 25, 1992
Volume
267
Issue
21
Pages
15032–15040
Identifiers
PMID: 1634539
Source
Medline
License
Unknown

Abstract

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.

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