Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii.
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.
Shen
Yulong
Y
Biological Information Research Center, National Institute of Advanced Science and Technology, Tsukuba, Ibaraki 305, Japan.
Tang
Xiao-Feng
XF
Matsui
Ikuo
I
D000595:Amino Acid Sequence; D003461:Crystallography; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D019711:Pyrococcus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D016335:Zinc Fingers / Q000502:physiology
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases