Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells
Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells
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DOI：
10.1016/j.yexcr.2007.04.003
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发表时间：
2007-07-15
影响因子：
3.7
通讯作者：
Karlsson, Anna
中科院分区：
文献类型：
--
作者：
Franco, Maribel;Johansson, Magnus;Karlsson, Anna
线粒体DNA复制所需的嘌呤脱氧核糖核苷酸要么从胞质溶胶输入，要么来源于线粒体脱氧鸟苷激酶（DGUOK）催化的脱氧腺苷或脱氧鸟苷的磷酸化。DGUOK缺乏与线粒体DNA耗竭综合征有关，表明这种酶在dNTP供应中起重要作用。我们通过表达针对DGUOK mRNA的小干扰RNA产生了DGUOK mRNA水平降低20-30%的HeLa细胞系。当在培养物中以指数方式生长时，酶表达降低的细胞显示出与对照细胞相似的mtDNA水平。然而，mtDNA水平迅速下降时，细胞周期停滞诱导血清饥饿。脱氧鸟苷激酶表达降低的细胞中9-（-D-阿拉伯呋喃糖基鸟嘌呤（araG）的DNA掺入较低，但细胞中araG磷酸化的总速率增加。araG磷酸化的增加被证明是由于脱氧胞苷激酶的表达增加。总之，我们的研究结果表明，DGUOK是在静息细胞中线粒体DNA复制所必需的，并且这种酶表达的微小变化可能导致线粒体DNA耗尽。我们的数据还表明，DGUOK表达水平的改变可能会诱导其他核苷激酶表达的代偿性变化。(C)2007爱思唯尔公司All rights reserved.
Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-(-D-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases. (C) 2007 Elsevier Inc. All rights reserved.