Characterization of human DNA polymerase delta and its subassemblies reconstituted by expression in the MultiBac system.
Characterization of human DNA polymerase delta and its subassemblies reconstituted by expression in the MultiBac system.
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DOI：
10.1371/journal.pone.0039156
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lee MY
中科院分区：
文献类型：
--
作者：
Zhou Y;Meng X;Zhang S;Lee EY;Lee MY
哺乳动物 DNA 聚合酶 δ (Pol δ) 是一种四亚基酶，在 DNA 复制和 DNA 修复过程中发挥着至关重要且多用途的作用。我们在感染单一杆状病毒的昆虫细胞中重建了人类 Pol δ 复合物，其中组装了一个或多个亚基。该系统允许四聚体Polδ全酶、p125/p50核心二聚体、核心+p68三聚体和核心+p12三聚体以及p125催化亚基的有效表达。通过高度标准化的方案，以毫克量分离出这些物质，具有可重复的纯度和特定的活性。我们系统地比较了它们的活动，以便深入了解 p12 和 p68 亚基的作用以及它们对 PCNA 的反应。 Pol δ 全酶、core+p68、core+p12 和 p125/p50 core 的相对比活性（表观 k cat）分别为 100、109、40 和 29。PCNA 相应的表观 K d 分别为 7.1、8.7、9.3 和 73 nM。我们的结果支持这样的假设：Pol δ 通过多种相互作用与 PCNA 相互作用，并且结合相互作用可能存在冗余，这可能允许 Pol δ 与 PCNA 采取灵活的配置。检查了 Pol δ 复合物完全延伸单引物 M13 DNA 的能力。除 core+p68 之外的所有亚基在完全延伸引物的能力上都有缺陷，表明 p68 亚基在该测定中在长 DNA 片段的合成中具有重要功能。核心+p68三聚体可以通过添加p12来重构。
Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent K d's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.
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