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Structural studies of the mechanism of clamp loading by clamp loader complexes.

机译：夹具加载器配合物对夹具加载机理的结构研究。

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摘要

High-speed DNA replication is an intricate process that requires the coordinated efforts of many proteins at the replication fork. The replicative DNA polymerases require tethering to the DNA substrate in order to remain bound to the template and replicate the DNA processively. The polymerases are tethered to DNA by attachment to ring-shaped processivity factors, known as sliding clamps, which encircle DNA. Pentameric molecular machines comprised of AAA+ subunits, known as clamp loaders, are required to link the sliding clamps to DNA topologically. Clamp loaders, through the binding and hydrolysis of ATP, catalyze the opening of the ring-shaped sliding clamps and the placement of the clamps around DNA. Interaction with a sliding clamp requires that the subunits of a clamp loader be loaded with ATP. Once bound to the clamp, the clamp loader complex binds to a primer-template junction, in the process threading the DNA through the open interface of the clamp. Binding to the primer-template junction induces a conformational change in the clamp loader that acts as a switch, activating the ATPase activity of the AAA+ subunits and resulting in release of the clamp and DNA by the clamp loader. The structural mechanisms by which clamp loaders recognize primer-template junctions and hydrolyze ATP in response to DNA binding are not well understood. In this dissertation, I report the crystal structure of the E. coli clamp loader, gamma complex, bound to primer-template DNA. The structure reveals that, when bound to DNA, the AAA+ domains of the clamp loader subunits adopt a highly symmetric spiral conformation that interacts with the helical DNA duplex, with the N-terminal domains of the subunits tracking the template strand of the primer-template junction. In this conformation, the ATP binding sites, which are formed at subunit-subunit interfaces within the AAA+ spiral, are all in the same ATPase activated conformation, suggesting a mechanism by which DNA binding promotes this conformation and thereby leads to ATP hydrolysis. An unexpected feature of this structure is that primer-template recognition is restricted primarily to the template strand, with virtually no contacts made with the primer strand. As a consequence of this mode of DNA binding in which contacts are restricted to the template strand, models for the recognition of RNA-DNA primer-template junctions, as well as the recognition of reverse polarity primer-templates, are proposed. A related structure which I also present, that of the E. coli clamp loader bound to DNA as well as a peptide derived from the N-terminal tail of the psi protein, a clamp loader binding partner, suggests a mechanism whereby the binding of the psi protein promotes the clamp and DNA binding activities of the clamp loader. Binding of this peptide promotes a conformational change within the collar domains of the clamp loader that is necessary for the clamp loader to adopt the highly symmetric spiral conformation of the AAA+ domains when bound to DNA.
机译：高速DNA复制是一个复杂的过程，需要许多蛋白质在复制叉处共同努力。复制性DNA聚合酶需要束缚在DNA底物上，以保持与模板的结合并逐步复制DNA。聚合酶通过附着在环绕DNA的环形滑动因子（称为滑动夹具）上而与DNA相连。需要由AAA +亚基组成的五聚体分子机器，称为钳夹加载器，才能将滑动钳与拓扑结构链接到DNA。夹具装载器通过ATP的结合和水解，催化环形滑动夹具的打开以及DNA周围夹具的放置。与滑动夹具的相互作用要求夹具加载器的子单元加载有ATP。一旦与夹具结合，夹具加载器复合物就结合到引物-模板连接处，在此过程中将DNA穿过夹具的开放界面。与引物-模板接合处的结合在夹具装载器中诱导构象变化，该构象变化充当开关，激活AAA +亚基的ATPase活性，并导致夹具装载器和DNA释放。钳加载器识别DNA结合反应的引物-模板连接并水解ATP的结构机理尚不清楚。在这篇论文中，我报道了结合到引物-模板DNA上的大肠杆菌钳形装载物γ复合物的晶体结构。该结构揭示，当与DNA结合时，钳位加载子亚基的AAA +域采用高度对称的螺旋构象，该构象与螺旋DNA双链体相互作用，且亚基的N末端域追踪引物模板的模板链交界处。在这种构象中，在AAA +螺旋内亚基-亚基界面处形成的ATP结合位点均处于同一ATPase激活的构象中，表明DNA结合促进该构象并导致ATP水解的机制。该结构的一个出乎意料的特征是，引物-模板识别主要限于模板链，而实际上与引物链没有接触。由于这种接触仅限于模板链的DNA结合方式，提出了用于识别RNA-DNA引物-模板连接以及识别反向极性引物-模板的模型。我还介绍了一个相关结构，即与DNA结合的大肠埃希菌钳状装载物以及从psi蛋白的N末端尾部衍生的肽（钳状装载物结合伴侣），提示了一种机制psi蛋白可促进钳夹装载器的钳夹和DNA结合活性。该肽的结合促进了钳夹装载器的环结构域内的构象变化，这对于钳夹装载器在与DNA结合时采用AAA +结构域的高度对称螺旋构象是必需的。

著录项

  • 作者

    Simonetta, Kyle Robert.;

  • 作者单位

    University of California, Berkeley.;

  • 授予单位 University of California, Berkeley.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 81 p.
  • 总页数 81
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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