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Plant and Cell Physiology Advance Access published online on October 17, 2007
Plant and Cell Physiology, doi:10.1093/pcp/pcm140
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

NtPolI-like1 and NtPolI-like2, bacterial DNA polymerase I homologues isolated from BY-2 cultured tobacco cells, encode DNA polymerases engaged in DNA replication in both plastids and mitochondria

Yuriko Ono1, Atsushi Sakai2, Katsuaki Takechi1, Susumu Takio3, Mari Takusagawa4 and Hiroyoshi Takano1

1Graduate School of Science and Technology, Kumamoto University Kumamoto 860-8555, Japan
2Department of Biological Sciences, Faculty of Science, Nara Women's University Nara 630-8506, Japan
3Center for Marine Environment Studies, Kumamoto University Kumamoto 860-8555, Japan
4Graduate School of Humanities and Sciences, Nara Women's University Nara 630-8506, Japan
Y. O. and A. S. contributed equally to this work.

Corresponding author: Atsushi Sakai, Department of Biological Sciences, Faculty of Science, Nara Women's University, Kitauoyanishi-machi, Nara 630-8506, Japan, Tel/Fax: +81-742-20-3425, E-mail: sakai{at}cc.nara-wu.ac.jp


  Abstract

Two cDNAs encoding homologues of bacterial DNA polymerase I were isolated from cultured tobacco (Nicotiana tabacum) BY-2 cells, and the corresponding genes were named NtPolI-like1 and NtPolI-like2. High sequence similarity suggested that they are orthologous genes each derived from respective parental species of N. tabacum, an allotetraploid plant. Each of the NtPolI-like1/2 gene products had a putative transit peptide for plastid localization at the N-terminus, followed by a 3'-5' exonuclease domain in the internal region, and a DNA polymerase domain in the C-terminal region. Among family A DNA polymerases, NtPolI-like proteins formed, together with other plant DNA polymerase I-homologues, a phylogenetic group distinct from mitochondrial DNA polymerase {gamma} in animals and fungi, as well as eukaryotic cell-nuclear localized repair enzymes. In contrast to computer predictions, experiments with GFP-fusion protein and western-blotting analysis suggested dual targeting of the gene products to both plastids and mitochondria. The recombinant NtPolI-like2 protein exhibited DNA polymerase activity in vitro. Their biochemical character roughly coincided with those of the 116-kDa DNA polymerases found in the plastid- and mitochondrial-nuclei (nucleoids) isolated from BY-2 cells. Pretreatment of the organelle-nuclear extracts with anti-NtPolI-like antibody removed most of the DNA polymerase activity. RT-PCR and western-blotting analyses demonstrated transient activation of NtPolI-like gene expression in the initial phase of cell proliferation, exactly when the 116-kDa DNAPs in the isolated organelle-nuclei were activated and preferential synthesis of organelle DNAs occurred. Taken together, our results suggest that NtPolI-like1/2 genes encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

Keywords: BY-2 - DNA polymerase - dual targeting - mitochondria - plastids - tobacco

(Received August 20, 2007; Accepted October 12, 2007)
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