Assembly and Distributive Action of an Archaeal DNA Polymerase Holoenzyme
Assembly and Distributive Action of an Archaeal DNA Polymerase Holoenzyme
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DOI：
10.1016/j.jmb.2013.09.003
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发表时间：
2013-11-29
影响因子：
5.6
通讯作者：
Trakselis, Michael A.
中科院分区：
文献类型：
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作者：
Bauer, Robert J.;Wolff, Ian D.;Trakselis, Michael A.
采用稳态荧光共振能量转移技术结合功能和结构研究，研究了硫磺硫化叶菌（Sulfolobus solfataricus，Sso）复制型DNA聚合酶全酶的组装和酶促能力。动力学实验表明，ATP与复制因子C（RFC）的结合足以将异源三聚体PCNA 123 [增殖细胞核抗原（PCNA）]夹加载到DNA上，其中包括复合物的限速构象重排。ATP水解是有利的招聘和与复制聚合酶（PolB 1）的相互作用，最有可能包括钳关闭和RFC解离所必需的。令人惊讶的是，组装的全酶复合物合成DNA的分布和低的持续合成能力，不像大多数其他良好表征的DNA聚合酶全酶复合物。我们发现PolB 1反复从DNA模板中脱离，留下PCNA 123。与PolB 1上新鉴定的C-末端PCNA相互作用肽（PIP）基序的相互作用特别是与PCNA 2的相互作用是全酶形成和合成过程中连续重新招募所必需的。在PolB 1中的C-末端PIP基序的延伸的尾状结构被单独揭示，并且当使用小角X射线散射与DNA结合时，使我们能够开发全酶复合物的模型。这是第一个详细的动力学描述的钳加载和全酶组装在crenarchaea，并揭示了一种新的模式，动态的持续合成能力发生的聚合酶交换机制。这项工作具有重要意义的进行性DNA复制合成，也表明了一个潜在的机制，聚合酶切换到旁路病变。(C)2013爱思唯尔有限公司保留所有权利。
The assembly and enzymatic ability of the replication DNA polymerase holoenzyme from Sulfolobus solfataricus (Sso) was investigated using presteady-state fluorescence resonance energy transfer assays coupled with functional and structural studies. Kinetic experiments reveal that ATP binding to replication factor C (RFC) is sufficient for loading the heterotrimeric PCNA123 [proliferating cell nuclear antigen (PCNA)] clamp onto DNA that includes a rate-limiting conformational rearrangement of the complex. ATP hydrolysis is required for favorable recruitment and interactions with the replication polymerase (PolB1) that most likely include clamp closing and RFC dissociation. Surprisingly, the assembled holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. We show that PolB1 repeatedly disengages from the DNA template, leaving PCNA123 behind. Interactions with a newly identified C-terminal PCNA-interacting peptide (PIP) motif on PolB1 specifically with PCNA2 are required for holoenzyme formation and continuous re-recruitment during synthesis. The extended tail-like structure of the C-terminal PIP motif in PolB1 is revealed alone and when bound to DNA using small-angle X-ray scattering allowing us to develop a model for the holoenzyme complex. This is the first detailed kinetic description of clamp loading and holoenzyme assembly in crenarchaea and has revealed a novel mode for dynamic processivity that occurs by a polymerase exchange mechanism. This work has important implications for processive DNA replication synthesis and also suggests a potential mechanism for polymerase switching to bypass lesions. (C) 2013 Elsevier Ltd. All rights reserved.