The Dynamics of Replication Processivity Factors

摘要：DNA复制是所有生物复制自身DNA的过程，是生物遗传的基础。新DNA链的合成是由一种名为DNA聚合酶的酶催化的，它使用母DNA链作为模板来合成新的副本。从细菌到人类，有效的DNA复制需要被称为加工性因子的蛋白质，以确保聚合酶沿着DNA快速移动，而不是与DNA分离。特别是，滑动夹是环绕DNA的低聚环状蛋白质，为DNA聚合酶提供锚。要将夹子加载到DNA上，必须形成一个开放的夹子加载器-夹子复合体。通常假设夹具在溶液中以闭合环的形式存在，因此夹具装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。我们将研究夹子打开的动力学，目的是了解夹子加载器能够将滑动夹子加载到DNA上的机制。从结构数据中出现的钳制负荷的相当静态的观点，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，该实验设计基于对少量分子的自发涨落的测量和分析。首先，将表征大肠杆菌(二聚体)和酿酒酵母(三聚体)的加工性钳的溶液齐聚平衡动力学。这些蛋白质是研究最多的滑动夹之一，然而它们的结合亲和力和速率常数还没有完全被表征。然后，将表征滑动夹在溶液中、结合到钳夹加载器和结合到DNA上的构象动力学。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，并将在这个项目中用于表征加工性钳制中的构象波动。这些研究的成功完成将为过程性因素是如何起作用的提供重要的机械性见解。更广泛的影响：研究生可以通过了解和参与莱维图斯(亚利桑那州立大学)和布鲁姆(UF)实验室的协同和共同努力的所有方面来丰富他们的教育经验。亚利桑那州立大学的学生将在每个暑假的一小部分时间里沉浸在该项目的分子生物学方面，而亚利桑那州立大学的学生将花费时间在亚利桑那州立大学学习单分子和其他光谱技术。亚利桑那州立大学和密歇根大学将通过一系列现有项目招收来自代表性不足群体的学生。将继续开展一系列旨在增加少数群体学生和职业生涯初期教师的留校率和成功机会的活动，包括指导女性初级教师和少数群体研究生，以及参加在STEM学科中任职人数不足的本科生的学生研究会议。

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.