复制体的冷冻电镜结构揭示了协调DNA合成的多种相互作用。
Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis.
作者信息
Kulczyk Arkadiusz W, Moeller Arne, Meyer Peter, Sliz Piotr, Richardson Charles C
机构信息
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
Department of Molecular Biology and Genetics, The Danish Research Institute of Translational Neuroscience, Aarhus University, 8000 Aarhus, Denmark.
出版信息
Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):E1848-E1856. doi: 10.1073/pnas.1701252114. Epub 2017 Feb 21.
Abstract
We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.
摘要
我们展示了噬菌体T7在类似复制叉的DNA上组装的约650 kDa功能性复制体的结构。通过单颗粒冷冻电子显微镜确定了由DNA解旋酶的六个结构域、RNA引发酶的五个结构域、两个DNA聚合酶和两个硫氧还蛋白（持续性因子）分子组成的复合物的结构。两个DNA聚合酶分子在复制叉处采用不同的空间排列，反映了它们在前导链和滞后链合成中的作用。该结构与生化数据相结合，揭示了前导链和滞后链合成协调的分子机制。由于DNA复制机制高度保守，这些观察结果与其他复制系统相关。
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Structure. 2017 Jan 3;25(1):157-166. doi: 10.1016/j.str.2016.11.019.
2
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3
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4
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5
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6
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Nat Struct Mol Biol. 2015 Dec;22(12):976-82. doi: 10.1038/nsmb.3113. Epub 2015 Nov 2.
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