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首页> 外文期刊>Journal of Molecular Biology >The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum.
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The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum.

机译：疟疾寄生虫恶性疟原虫的原生质体基因组复制DNA聚合酶Pfprex合成的生物化学和保真度。

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摘要

Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an approximately 35-kb circular DNA genome of unusually high A/T content (>86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3'-->5' exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T-->C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.
机译：恶性疟原虫是人类疟疾的主要病原体，包含三个独立的基因组。 apicoplast（细胞内细胞器）包含一个大约35 kb的环状DNA基因组，具有异常高的A / T含量（> 86％），该基因组由核编码的复制复合物Pfprex复制。在此，我们已经表达和纯化了Pfprex [KPom1（疟疾1的Klenow样聚合酶）]的DNA聚合酶结构域，并使用基于LacZ的正向突变测定法测量了其保真度。此外，我们分析了使用缺乏3'-> 5'外切核酸活性的Kpom1结合互补和非互补核苷酸的动力学参数。 KPom1表现出强烈偏向的突变谱，其中T-> C是最常见的单碱基取代，与密切相关的大肠杆菌DNA聚合酶I明显不同。我们使用具有温度敏感性聚合酶I等位基因的大肠杆菌建立了KPom1可以在高温下补充生长缺陷表型。我们建议可以在互补测定法中利用KPom1的错误偏倚来识别可模仿这种碱基错配并优先抑制apicoplast DNA复制的核苷类似物。

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