Plant and Cell Physiology Advance Access originally published online on October 17, 2007
Plant and Cell Physiology 2007 48(12):1679-1692; doi:10.1093/pcp/pcm140
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NtPolI-like1 and NtPolI-like2, Bacterial DNA Polymerase I Homologs Isolated from BY-2 Cultured Tobacco Cells, Encode DNA Polymerases Engaged in DNA Replication in Both Plastids and Mitochondria
1 Graduate School of Science and Technology, Kumamoto University, Kumamoto, 860-8555 Japan
2 Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara, 630-8506 Japan
3 Center for Marine Environment Studies, Kumamoto University, Kumamoto, 860-8555 Japan
4 Graduate School of Humanities and Sciences, Nara Women's University, Nara, 630-8506 Japan
*Corresponding author: E-mail, sakai{at}cc.nara-wu.ac.jp; Fax, +81-742-20-3425.
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Abstract |
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Two cDNAs encoding homologs 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 homologs, a phylogenetic group distinct from mitochondrial DNA polymerase 
in animals and fungi, as well as eukaryotic cell nuclear-localized repair enzymes. In contrast to computer predictions, experiments with green fluorescent protein (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. Pre-treatment of the organelle nuclear extracts with anti-NtPolI-like antibody removed most of the DNA polymerase activity. Reverse transcription–PCR (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 DNA polymerases 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
Abbreviations: CaMV, cauliflower mosaic virus; ddNTP, dideoxyribonucleoside triphosphate; DNAP, DNA polymerase; DTT, dithiothreitol; EST, expressed sequence tag; GFP, green fluorescent protein; IPTG, isopropyl-β-D-thiogalactopyranoside; NEM, N-ethylmaleimide; PolI, DNA polymerase I of E. coli; RT–PCR, reverse transcription–PCR; RACE, rapid amplification of cDNA ends
5These authors contributed equally to this work.
(Received August 20, 2007; Accepted October 12, 2007)
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