The Dynamics of Replication Processivity Factors

复制过程因素的动态变化

• 批准号: 1157765
• 负责人: Marcia Levitus
• 金额: $ 48.14万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1157765&HistoricalAwards=false
• 关键词: Dynamics; Replication; Processivity; Factors

AbstractIntellectual Merit: DNA replication is the process by which all living organisms make copies of their DNA, and it is the foundation of biological inheritance. The synthesis of the new DNA strands is catalyzed by enzymes called DNA polymerases, which use the mother DNA strand as a template to synthesize a new copy. From bacteria to humans, efficient DNA replication requires proteins known as processivity factors to ensure that the polymerase moves rapidly along DNA without dissociating from it. In particular, sliding clamps are oligomeric ring-shaped proteins that encircle DNA, providing an anchor for the DNA polymerase. To load clamps onto DNA, an open clamp loader-clamp complex must form. It is generally assumed that clamps exist as closed rings in solution and that clamp loaders must therefore actively open their interfaces. Very few studies, however, have addressed this problem directly. The dynamics of clamp opening will be investigated with the goal of understanding the mechanisms by which clamp loaders are able to load sliding clamps onto DNA. The fairly static view of clamp loading that has emerged from structural data is intrinsically inadequate to understand the mechanistic details of how these proteins achieve their function. This limitation will be tackled directly by our experimental design, which is based on the measurement and analysis of the spontaneous fluctuations of a small number of molecules. Initially, the solution oligomerization equilibrium dynamics of the processivity clamps of E. coli (a dimer) and S. cerevisiae (a trimer) will be characterized. These proteins are among the most studied sliding clamps, and yet their association affinities and rate constants have not been fully characterized. Then, the conformational dynamics of sliding clamps in solution, bound to the clamp loaders, and bound to DNA will be characterized. Single-molecule fluorescence techniques are particularly well-suited to investigate the structural dynamics of biopolymers, and will be used in this project to characterize the conformational fluctuations in processivity clamps. The successful completion of these studies will provide vital mechanistic insights into how processivity factors work. Broader Impacts: Graduate students can enrich their educational experience by learning about and participating in all aspects of the synergistic and joint efforts of the Levitus (ASU) and Bloom (UF) labs. ASU students will spend a fraction of each summer at UF to immerse themselves into the molecular biology aspects of the project, while a student from UF will spend time at ASU to learn about single-molecule and other spectroscopic techniques. Students from underrepresented groups will be recruited through a series of existing programs at ASU and UF. A series of activities aimed at increasing the retention and chances of success of minority students and early-career faculty, including mentoring female junior faculty and minority graduate students, will be continued, as well as participation in student research conferences for underrepresented undergraduate students within the STEM disciplines.

摘要智力价值：DNA复制是所有生物体复制其DNA的过程，它是生物遗传的基础。新DNA链的合成是由称为DNA聚合酶的酶催化的，该酶使用母亲DNA链作为模板来合成新的拷贝。从细菌到人类，有效的DNA复制都需要一种蛋白质，这种蛋白质被称为持续合成因子，以确保聚合酶沿着DNA快速沿着移动，而不会与DNA分离。特别是，滑动夹是一种寡聚环状蛋白质，它包围着DNA，为DNA聚合酶提供了一个锚。为了将钳加载到DNA上，必须形成开放钳加载器-钳复合物。通常假设夹具在溶液中作为闭合环存在，因此夹具加载器必须主动打开其接口。然而，很少有研究直接解决这个问题。将研究夹钳打开的动力学，目的是了解夹钳加载器能够将滑动夹钳加载到DNA上的机制。从结构数据中出现的钳负载的相当静态的观点本质上不足以理解这些蛋白质如何实现其功能的机制细节。我们的实验设计将直接解决这一限制，该设计基于对少量分子自发波动的测量和分析。 首先，研究了E. coli（二聚体）和S.酿酒酵母（三聚体）将被表征。这些蛋白质是研究最多的滑动钳之一，但它们的结合亲和力和速率常数尚未得到充分表征。然后，将表征溶液中的滑动夹、与夹加载器结合以及与DNA结合的构象动力学。单分子荧光技术特别适合于研究生物聚合物的结构动力学，并将在本项目中用于表征持续合成能力钳的构象波动。这些研究的成功完成将为持续合成能力因素如何工作提供重要的机理见解。更广泛的影响：研究生可以通过学习和参与Levitus（ASU）和Bloom（UF）实验室的协同和共同努力的各个方面来丰富他们的教育经验。亚利桑那州立大学的学生将花费每年夏天在UF的一小部分沉浸到该项目的分子生物学方面，而从UF的学生将花时间在亚利桑那州立大学学习单分子和其他光谱技术。来自代表性不足群体的学生将通过ASU和UF的一系列现有计划招募。将继续开展一系列旨在提高少数民族学生和早期职业教师的保留率和成功机会的活动，包括指导女初级教师和少数民族研究生，以及参加STEM学科内代表性不足的本科生的学生研究会议。