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首页> 外文期刊>Molecular and Cellular Biology >The Human DNA Ligase III Gene Encodes Nuclear and Mitochondrial Proteins
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The Human DNA Ligase III Gene Encodes Nuclear and Mitochondrial Proteins

机译：人类DNA连接酶III基因编码核和线粒体蛋白。

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We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.
机译：我们提供证据表明人类DNA连接酶III基因编码该酶的线粒体形式。首先，DNA连接酶III cDNA包含一个框内ATG，位于假定的翻译起始起始位点上游。这两个ATG位点之间的DNA序列编码两亲性螺旋，类似于先前鉴定的线粒体靶向肽。第二，在其氨基末端具有该序列的重组绿色荧光蛋白被有效地靶向Cos-1猴肾细胞的线粒体。相反，天然绿色荧光蛋白分布在细胞质中。第三，将一系列血凝素-DNA连接酶III小基因构建体引入Cos-1细胞，并使用免疫细胞化学确定表位标记的DNA连接酶III蛋白的亚细胞定位。这些实验表明，上游ATG的失活导致DNA连接酶III蛋白的核积累，而下游ATG的失活则消除了核的定位，并导致线粒体区室中的积累。第四，从人细胞中过表达反义DNA连接酶III mRNA的线粒体蛋白提取物比从对照细胞制备的线粒体提取物具有更低的DNA连接酶活性。从反义mRNA表达细胞制备的提取物中，DNA末端连接活性也大大降低。从这些结果，我们得出结论，人类DNA连接酶III基因编码核酶和线粒体酶。 DNA连接酶在DNA复制，重组和DNA修复中起着核心作用。因此，这种酶的线粒体形式的鉴定提供了一种用于剖析哺乳动物线粒体基因组动力学的工具。

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