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
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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DOI：
10.1093/pcp/pcm140
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发表时间：
2007-12-01
影响因子：
4.9
通讯作者：
Takano, Hiroyoshi
中科院分区：
文献类型：
--
作者：
Ono, Yuriko;Sakai, Atsushi;Takano, Hiroyoshi
从培养的烟草（Nicotiana tabacum）BY-2细胞中分离到编码细菌DNA聚合酶I同源物的两个cDNA，相应的基因被命名为NtPolI-like1和NtPolI-like2。高度的序列相似性表明它们是直系同源基因，均源自异源四倍体植物烟草各自的亲本物种。每个 NtPolI-like1/2 基因产物在 N 末端都有一个用于质体定位的推定转运肽，随后在内部区域有一个 3'-5' 核酸外切酶结构域，在 C 末端区域有一个 DNA 聚合酶结构域。在 A 家族 DNA 聚合酶中，NtPolI 样蛋白与其他植物 DNA 聚合酶 I 同源物一起形成了一个不同于动物和真菌中线粒体 DNA 聚合酶 γ 的系统发育群，以及真核细胞核定位修复酶。与计算机预测相反，绿色荧光蛋白（GFP）融合蛋白和蛋白质印迹分析的实验表明基因产物双重靶向质体和线粒体。重组NtPolI-like2蛋白在体外表现出DNA聚合酶活性。它们的生化特征与从 BY-2 细胞中分离的质体和线粒体核（类核）中发现的 116kDa DNA 聚合酶的生化特征大致一致。用抗 NtPolI 样抗体对细胞器核提取物进行预处理，去除了大部分 DNA 聚合酶活性。逆转录 PCR (RT-PCR) 和蛋白质印迹分析表明，在细胞增殖的初始阶段，NtPolI 样基因表达瞬时激活，此时分离的细胞器核中的 116 kDa DNA 聚合酶被激活，细胞器 DNA 优先合成发生。综上所述，我们的结果表明 NtPolI-like1/2 基因编码 DNA 聚合酶，参与质体和线粒体中的 DNA 复制。
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 gamma 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 116kDa 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.