The Role of the DnaX Complex in Assembling Pol III Initation Complexes

描述（由申请人提供）：所有细胞在细胞分裂过程中必须快速准确地复制其DNA。DNA复制是由复杂的多蛋白组装进行的，这些蛋白质必须以高度协调的方式被招募到模板DNA上并装载到模板DNA上。来自大肠杆菌的DNA聚合酶III （Pol III）全酶是研究DNA复制的模型系统。Pol III全酶的一个关键成分是5亚基DnaX复合物，已知其功能是将“滑动钳”加工因子加载到DNA上，并协调前导链和滞后链的合成。其中一个DnaX亚基是DnaX基因的产物，在体内以全长或截短的形式存在。虽然这两种形式都支持DNA的过程合成，但这两种成分之间存在重要的功能差异。本提案的目标是确定两种形式的DnaX蛋白如何影响启动DNA复制的机制，从而更好地理解DnaX复合物的作用。将开发快速动力学分析来测量Pol III全酶在DNA模板上组装的速率。将使用KinTek快速混合/猝灭装置和荧光共振能量转移技术。全长DnaX蛋白的动力学优势将在实验中得到证实。通过考察每一种全酶组分浓度变化时的动力学效应，我们将详细阐述全酶/DNA起始复合物形成的机制。这些实验将验证核心Pol III DNA聚合酶被包含全长DnaX蛋白的DnaX复合物“陪伴”到起始复合物的假设。快速准确地复制遗传信息是细胞分裂的一个基本过程。因此，加深我们对这一过程的理解的研究对于开发更有效的治疗癌症或致病性感染的方法至关重要。这一建议的结果将确定DNA复制机制中关键组成部分的作用，并将导致对DNA复制如何在分子水平上启动的更好理解。

DESCRIPTION (provided by applicant): All cells must rapidly and accurately replicate their DNA during the process of cell division. DNA replication is carried out by complex multi-protein assemblies, which must be recruited to and loaded onto the template DNA in a highly coordinated fashion. The DNA polymerase III (Pol III) holoenzyme from the bacterium E. coli is a model system for studies of DNA replication. A key component of the Pol III holoenzyme is the 5 subunit DnaX complex, which has known functions in loading the "sliding clamp" processivity factor onto the DNA and in coordinating leading and lagging strand synthesis. One of the DnaX subunits, the product of the DnaX gene, occurs in vivo as either a full length or truncated version. While both forms support processive DNA synthesis, there are important functional differences between the two components. The goal of this proposal is to ascertain how the two forms of the DnaX protein affect the mechanism for initiating DNA replication and to thereby better understand the role of the DnaX complex. Rapid kinetic assays will be developed to measure the rate for assembling the Pol III holoenzyme on a DNA template. Both a KinTek rapid mix/quench device and fluorescence resonance energy transfer techniques will be utilized. The kinetic advantage of the full length DnaX protein over the truncated version will be established experimentally. The mechanism by which holoenzyme/DNA initiation complexes are formed will be elaborated by examining the kinetic effects as the concentration of each holoenzyme component is varied. These experiments will test the hypothesis that the core Pol III DNA polymerase is "chaperoned" to the initiation complex by DnaX complexes containing the full length DnaX protein. PUBLIC HEALTH RELEVANCE The fast and accurate replication of genetic information is a fundamental process of cell division. Thus, research that deepens our understanding of this process is crucial for developing more effective treatments for cancer or pathogenic infection. The results of this proposal will establish the role of a key component in the DNA replication machinery and will lead to a greater understanding of how DNA replication is initiated at the molecular level.