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首页> 外文学位 >The clamp loader of Escherichia coli DNA polymerase III: Kinetics of the ATP-dependent steps in the sliding -clamp loading reaction.
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The clamp loader of Escherichia coli DNA polymerase III: Kinetics of the ATP-dependent steps in the sliding -clamp loading reaction.

机译：大肠杆菌DNA聚合酶III的钳式装载器：滑动钳式装载反应中ATP依赖性步骤的动力学。

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摘要

DNA polymerase III holoenzyme, the principal enzyme responsible for E. coli chromosomal replication, synthesizes stretches of DNA thousands of nucleotides long at a rate approaching 750 nucleotides s-1 without dissociation. A ring-shaped DNA sliding-clamp "beta" topologically links the polymerase to DNA. A clamp loader, "gamma complex", assembles beta on DNA in an ATP-dependent reaction. This dissertation project was undertaken for investigation of the mechanism of beta clamp loading by the clamp loader for processive replication. ATP binding and hydrolysis activities of the clamp loader promote conformational changes modulating its binding affinity for the clamp and DNA. Using fluorescence-based steady-state and real-time stopped-flow methods, the kinetics of these dynamic conformational changes were measured. Pre-steady-state ATP hydrolysis assays performed in the absence or presence of beta resulted in biphasic or monophasic kinetics respectively. Biphasic kinetics suggested that the clamp loader, equilibrated with ATP, exists in a mixture of two dominant species. Addition of beta converted this mixture into a single activated population, effectively increasing its concentration. These experiments, in addition to adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS)-chase assays showed that activated gamma complex hydrolyzed one ATP per gamma-subunit at a rate faster than ATP dissociation. The hypothesis that gamma complex exists in multiple species with ATP was confirmed by measuring pre-steady-state clamp loading kinetics in DNA-binding assays. When gamma complex was equilibrated with ATP, a mixture of two species formed which when mixed with DNA and beta exhibited biphasic DNA binding kinetics. When equilibrated with ATP and beta, rapid monophasic DNA binding kinetics resulted. Direct mixing of gamma complex with DNA beta and ATP displayed slow monophasic kinetics, limited by the conformational change rate. The rate of the conformational changes separating the two dominant species was determined by investigating their evolution in equilibration time with ATP. Computer modeling of these experimental data revealed a conformational change rate of ∼4.5 s-1. Comparison of the kinetics of a "minimal" clamp loader complex missing chi and psi subunits, revealed that these subunits facilitate the conformational changes in gamma complex required for modulation of beta and DNA binding affinities during the clamp loading reaction.
机译：DNA聚合酶III全酶，是负责大肠杆菌染色体复制的主要酶，可以合成DNA数千个核苷酸的片段，其长度接近750个核苷酸s-1，而不会解离。环状DNA滑动夹“β”在拓扑上将聚合酶连接到DNA。钳式装载机，“伽马复合物”，以ATP依赖的反应在DNA上组装β。本论文的目的是为了研究β-钳夹通过钳式装载机进行连续复制的机理。钳装载器的ATP结合和水解活性促进构象变化，从而调节其对钳和DNA的结合亲和力。使用基于荧光的稳态和实时停止流方法，测量了这些动态构象变化的动力学。在不存在或存在β的情况下进行的稳态前ATP水解测定分别导致了双相或单相动力学。双相动力学表明，与ATP平衡的钳式装载器以两种主要物质的混合物形式存在。添加β会将这种混合物转化为单个活化的种群，从而有效地提高了其浓度。这些实验，除了腺苷5'-O-（3-硫代三磷酸）（ATPgammaS）-追逐测定法显示，活化的γ-复合物以比ATP解离更快的速率水解每个γ-亚基一个ATP。通过在DNA结合测定中测量稳态前钳位加载动力学，证实了γ络合物存在于多种ATP中的假说。当用ATP平衡γ复合物时，形成了两种物质的混合物，当与DNA和β混合时，它们表现出双相DNA结合动力学。当与ATP和β平衡时，可产生快速的单相DNA结合动力学。 γ复合物与DNAβ和ATP的直接混合显示出缓慢的单相动力学，受构象变化率的限制。分离两个优势种的构象变化率是通过研究ATP在平衡时间内的进化来确定的。这些实验数据的计算机建模显示构象变化率为约4.5 s-1。缺少chi和psi亚基的“最小”夹具装载物复合物的动力学比较表明，这些亚基促进了在夹具装载反应过程中调节β和DNA结合亲和力所需的γ复合物构象变化。

著录项

  • 作者

    Williams, Christopher R.;

  • 作者单位

    University of Florida.;

  • 授予单位 University of Florida.;
  • 学科 Chemistry Biochemistry.;Biophysics General.;Biology Molecular.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 290 p.
  • 总页数 290
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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