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J. Biol. Chem., Vol. 278, Issue 23, 21247-21257, June 6, 2003

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Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii^*,

Yulong Shen, Xiao-Feng Tang and Ikuo Matsui 

From the Biological Information Research Center, National Institute of Advanced Science and Technology, Tsukuba, Ibaraki 305, Japan

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.

View larger version (44K): [in this window] [in a new window]   FIG. 1. Schematic maps and the phenotypes summarized of the mutants analyzed in this study. Vectors are named as in Table I, as indicated with Footnote a. Footnote b shows the conserved region among DP1s that shows homology to the eukaryote small subunit of DNA polymerase is indicated in the box colored in yellow. The mini-intein insertion site and the catalytic center for polymerization in DP2 are shown with arrows and vertical lines. The region that shows homology to the catalytic subunit of yeast DNA polymerase in DP2 is indicated in the box colored in red. Two cysteine clusters are indicated with boxes filled with horizontal lines. The numbers in bold and blue in the parentheses are the numbers of peptide without the intein. Footnote c indicates the thermostability of the truncated peptide (ther. stab.), defined as the presence (+) or absence (-) of specific bands on SDS-GAGE analysis of the supernatant after heating at 85 °C for 30 min (Fig. 3). Subunit Inter., subunit interaction, the ability of complex formation between DP1Pho and DP2Pho after purification steps; Pol., polymerization; Exo., 3'-5' exonuclease activities; +, positive; -, negative; blank, not checked.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Alignment of the homologous regions of DP2Pho and the large subunit of DNA polymerase of S. cerevisiae. The region was identified when the DNA data bases were searched by BLAST using DP2Pho (without the intein) as a query. Invariant and conserved residues between DP2 from the 12 species and yeast DNA polymerase are in red and blue, respectively. Symbols + and the letters between the sequences indicate conserved and identical residues. The residue numbers of PolDPho are those in which the intein is included.

Received for publication, December 3, 2002 , and in revised form, March 20, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. 1 and 2.

To whom correspondence should be addressed: Biological Information Research Center, National Institute of Advanced Science and Technology, Tsukuba, Ibaraki 305-8566, Japan. Tel.: 81-298-616142; Fax: 81-298-616151; E-mail: ik-matsui{at}aist.go.jp.

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