Journal Article

<i>NtPolI-like1</i> and <i>NtPolI-like2</i>, Bacterial DNA Polymerase I Homologs Isolated from BY-2 Cultured Tobacco Cells, Encode DNA Polymerases Engaged in DNA Replication in Both Plastids and Mitochondria

Yuriko Ono, Atsushi Sakai, Katsuaki Takechi, Susumu Takio, Mari Takusagawa and Hiroyoshi Takano

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 48, issue 12, pages 1679-1692
Published in print December 2007 | ISSN: 0032-0781
Published online December 2007 | e-ISSN: 1471-9053 | DOI: https://dx.doi.org/10.1093/pcp/pcm140
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

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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 γ 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

Journal Article.  8754 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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