Family D DNA polymerase (PolD) has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. We successfully cloned, expressed, and purified the family D DNA polymerase from Pyrococcus horikoshii (PolDPho). By site-directed mutagenesis, we identified amino acid residues Asp-1122 and Asp-1124 of a large subunit as the essential residues responsible for DNA-polymerizing activity. We analysed the domain structure using proteins truncated at the N- and C-termini of both small and large subunits (DP1Pho and DP2Pho), and identified putative regions responsible for subunit interaction, oligomerization and regulation of the 3´-5´ exonuclease activity in PolDPho. It was also found that the internal region of the putative zinc finger motif (cysteine cluster II) at the C-terminal of DP2Pho is involved in the 3´-5´ exonuclease activity. Using gel filtration analysis, we determined the molecular masses of the recombinant PolDPho and the N-terminal putative dimerization domain of the large subunit, and proposed that PolD from P. horikoshii probably forms a heterotetrameric structure in solution. Based on these results, a model regarding the subunit interaction and regulation of activity of PolDPho is proposed.
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April 2004
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Conference Article|
April 01 2004
Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii
Y. Shen;
Y. Shen
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
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X.-F. Tang;
X.-F. Tang
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
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E. Matsui;
E. Matsui
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
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I. Matsui
I. Matsui
1
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
1To whom correspondence should be addressed (e-mail ik-matsui@aist.go.jp).
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Publisher: Portland Press Ltd
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2004 Biochemical Society
2004
Biochem Soc Trans (2004) 32 (2): 245–249.
Citation
Y. Shen, X.-F. Tang, E. Matsui, I. Matsui; Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii. Biochem Soc Trans 1 April 2004; 32 (2): 245–249. doi: https://doi.org/10.1042/bst0320245
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