Structural and functional organization of the DNA polymerase of bacteriophage T7

J Biol Chem. 1996 Sep 27;271(39):24207-12. doi: 10.1074/jbc.271.39.24207.

Abstract

The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / ultrastructure
  • Exonucleases / chemistry
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Protein Binding
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Thioredoxins / metabolism*
  • Viral Proteins / chemistry

Substances

  • Bacterial Proteins
  • Peptide Fragments
  • Viral Proteins
  • Thioredoxins
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase
  • Exonucleases