Identification of a DNA primase template tracking site redefines the geometry of primer synthesis
Identification of a DNA primase template tracking site redefines the geometry of primer synthesis
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DOI：
10.1038/nsmb.1373
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发表时间：
2008-02-01
影响因子：
16.8
通讯作者：
Berger, James M.
中科院分区：
文献类型：
--
作者：
Corn, Jacob E.;Pelton, Jeffrey G.;Berger, James M.
引发酶是DNA复制起始、后随链合成以及复制重启所必需的重要RNA聚合酶。引发酶功能的许多方面仍不清楚，包括该酶如何与从解旋酶延伸出的移动核酸链结合，以及如何定向引物以交接给复制体组分。我们利用一种新的筛选方法来捕获瞬时大分子相互作用，确定了大肠杆菌DnaG引发酶催化结构域与单链DNA结合的结构。该结构揭示了一个意想不到的结合位点，它以两种不同的构象与核酸结合，表明它作为模板DNA的非特异性捕获和追踪位点。生物信息学和生物化学分析表明，这个在进化上受约束的区域在活性位点附近强化了模板极性，并且是引发酶功能所必需的。总之，我们的研究结果推翻了先前关于引物 - 模板定向的观点，并调和了不同的研究以重新评估复制叉的组织。
Primases are essential RNA polymerases required for the initiation of DNA replication, lagging strand synthesis and replication restart. Many aspects of primase function remain unclear, including how the enzyme associates with a moving nucleic acid strand emanating from a helicase and orients primers for handoff to replisomal components. Using a new screening method to trap transient macromolecular interactions, we determined the structure of the Escherichia coli DnaG primase catalytic domain bound to single-stranded DNA. The structure reveals an unanticipated binding site that engages nucleic acid in two distinct configurations, indicating that it serves as a nonspecific capture and tracking locus for template DNA. Bioinformatic and biochemical analyses show that this evolutionarily constrained region enforces template polarity near the active site and is required for primase function. Together, our findings reverse previous proposals for primer-template orientation and reconcile disparate studies to re-evaluate replication fork organization.