Polymerase coordination at the Pol III replication fork

描述（由申请人提供）：基因组信息的成功复制对细胞分裂和病毒繁殖至关重要。加深我们对基因组复制的理解的研究对于癌症以及细菌、寄生虫和病毒感染的治疗进展至关重要。大肠杆菌（E. coli）是研究DNA复制的模型系统，因为它易于处理和遗传操作，关于这种生物的大量信息，以及它与真核复制系统的许多机制相似性。大肠杆菌复制酶，DNA Pol III全酶，由10个不同的蛋白质亚基组成。虽然许多亚基的化学计量、位置和一般功能已经确定，但亚基之间动态相互作用的意义尚未得到充分理解。本文提出的研究目标是进一步阐明DNA Pol III复制酶内影响复制叉处前导链和滞后链聚合酶协调的通讯机制。重组的DNA复制系统将与BIAcore生物传感器和荧光各向异性技术一起使用，以识别在滞后链上发出聚合酶循环信号的效应物。将测量不同信号机制引起聚合酶与DNA分离的速率，以确定其生理相关性。此外，将使用一种新的滚圈复制系统来研究复制叉上两种聚合酶之间的通信，其中可以测量对前导链合成的影响，而滞后链聚合酶的行为是不同的。

DESCRIPTION (provided by applicant): The successful duplication of genomic information is essential for cell division and viral propagation. Research that deepens our understanding of genomic replication is crucial for advance in the treatment of cancer as well as infection by bacteria, parasites, and viruses. The bacterium Escherichia coli (E. coli) is a model system for studying DNA replication due to its ease of handling and genetic manipulation, the large amount of information available about this organism, and its many mechanistic similarities with eukaryotic replication systems. The E. coli replicase, DNA Pol III holoenzyme, is composed of 10 different protein subunits. While the stoichiometry, placement, and general function have been determined for many subunits, the significance of dynamic interactions between subunits has yet to be fully understood. The goal of the research proposed here is to further elucidate the communication mechanisms within the DNA Pol III replicase that affect coordination of the leading and lagging strand polymerases at the replication fork. A reconstituted DNA replication system will be used in conjunction with BIAcore biosensor and fluorescence anisotropy techniques to identify the effectors that signal polymerase cycling on the lagging strand. The rates at which different signaling mechanisms cause polymerase dissociation from DNA will be measured in order to determine their physiological relevance. Additionally, communication between the two polymerases at the replication fork will be studied using a novel rolling circle replication system in which the affects on leading strand synthesis can be measured while the behavior of the lagging strand polymerase is varied.