Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase
Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase
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DOI：
10.1074/jbc.m800310200
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发表时间：
2008-05-30
影响因子：
4.8
通讯作者：
Suo, Zucai
中科院分区：
文献类型：
--
作者：
Fowler, Jason D.;Brown, Jessica A.;Suo, Zucai
吉西他滨，2‘-脱氧-2’，2‘-二氟胞苷(DFDC)，是一种被批准用于治疗多种实体肿瘤的药物。临床上，这种中等毒性的核苷类似物会导致癌症患者的周围神经病变、血液功能障碍和肺部毒性。尽管这些副作用与线粒体功能障碍的症状非常相似，但没有直接证据表明吉西他滨干扰了由人类DNA聚合酶伽马催化的线粒体DNA复制。在这里，我们使用稳态态动力学方法直接研究了5‘-三磷酸化吉西他滨(DFdCTP)的掺入，掺入的单磷酸化形式(DFdCMP)的切除，以及人DNA聚合酶γ催化的模板碱基DFDC的旁路。与模板碱基dG相反，dFdCTP的掺入效率比dCTP低432倍。虽然DFDC不是链终止子，但被掺入的dFdCMP使接下来2个正确核苷酸的掺入效率分别降低了214倍和7倍。此外，3‘-dFdCMP的切割速度比匹配的3’-dCMP慢50倍。当遇到DFDC作为模板碱基时，DNA聚合酶伽马在病变和一个下游位置暂停，但最终将引物延长为全长产物。这些暂停是因为核苷酸掺入效率降低了1000倍。有趣的是，这些停顿部位的聚合酶保真度下降了2个数量级。因此，我们的稳态前动力学研究提供了直接证据证明吉西他滨对人线粒体DNA聚合酶活性的抑制作用。
Gemcitabine, 2'-deoxy-2',2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma. Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase gamma. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214- and 7-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase gamma paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.