Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii

J Biol Chem. 2003 Jun 6;278(23):21247-57. doi: 10.1074/jbc.M212286200. Epub 2003 Mar 24.


Functions of the terminal domains of the family D DNA polymerase from Pyrococcus horikoshii (PolDPho) were analyzed by making and characterizing various truncated proteins. Based on a co-expression vector developed previously (Shen, Y., Musti, K., Hiramoto, M., Kikuchi, H., Kawabayashi, Y., and Matsui, I. (2001) J. Biol. Chem. 276, 27376-27383), 25 vectors for terminal truncated proteins were constructed. The expressed proteins were characterized in terms of thermostability, subunit interaction, and polymerization and 3'-5' exonuclease activities. The carboxyl-terminal (1255-1332) of the large subunit (DP2Pho) and two regions, the 201-260 and 599-622, of the small subunit (DP1Pho) were found to be critical for the complex formation, and probable subunit interaction of PolDPho. The amino-terminal (1-300) of DP2Pho is essential for the folding of PolDPho and is likely the oligomerization domain of PolDPho. A short region at the extreme C-terminal of DP2Pho (from 1385 to 1434) and the N-terminal of DP1Pho(1-200), which forms a stable protein, are not absolutely necessary for either polymerization or the 3'-5' exonuclease activity. We identified a possible regulatory role of DP1Pho(1-200) for the 3'-5' exonuclease. Deletion of DP1Pho(1-200) increased the exonuclease and DNA binding activities of PolDPho. Adding DP1Pho(1-200) to the truncated protein suppressed the elevated exonuclease activity. We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3'-5' exonuclease, but is dispensable for the DNA polymerization.

MeSH terms

  • Amino Acid Sequence
  • Crystallography
  • DNA Primers
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Exonucleases / chemistry
  • Exonucleases / genetics
  • Exonucleases / metabolism
  • Gene Deletion
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Pyrococcus / enzymology*
  • Structure-Activity Relationship
  • Zinc Fingers / physiology


  • DNA Primers
  • DNA-Directed DNA Polymerase
  • Exonucleases