Mechanism of Termination of DNA Replication

描述(申请人提供)：本申请描述了实验 旨在解决关于以下机制的两个重要问题 原核生物中的复制分叉抑制和一系列广泛的实验 识别和研究参与复制的蛋白质的作用机制 在酵母rDNA中的终止，因此代表了工作从 从原核生物到真核生物。第一，有条件的，ppGpp依赖的机制 枯草芽孢杆菌复制检查点的叉子停滞将是 分析以调查是否由 复制终止蛋白(RTP)是将弱信号转化为 复制暂停站点到一个有效的终端。前面的实验将 利用荧光原位杂交、甲醛交联和 以DNA微阵列为主要实验工具。第二，机械论 大肠杆菌TUS和DNAB之间相互作用的方面将是 通过反向2-杂交分析鉴定解旋酶的突变 允许解旋酶穿过复制屏障的DNAB 终点站。这些突变体及其在Tus中的抑制子将与 用EM和3D图像研究TUS-DNAB相互作用的野生型蛋白质 重建。首席调查员试图解决的主要问题是 TUS和DNAB之间的接触是如何阻止解旋酶的 后者的活动，以及身体接触是否抑制 DNAB的DNA依赖的ATPase活性。一种新的基于复制激活的方法 将使用2-混合系统来识别Ter-Tus复合体或 DIF-XerC/D复合体与位于Z环或其附近的蛋白质相互作用 在染色体复制过程中，将Ter定位并保持在细胞中心 种族隔离。最后，涉及酵母遗传学和 分子生物学将被用来鉴定与fob相互作用的蛋白质。 1在酵母rDNA上逮捕叉子。首席调查员还将尝试 鉴定酵母复制装置中与之相互作用的成分 终结者情结。

DESCRIPTION (provided by applicant): This application describes experiments designed to address two significant questions regarding the mechanism of replication fork arrest in prokaryotes and an extensive set of experiments to identify and study the mechanism of action of proteins involved in replication termination in yeast rDNA and thus represents a transition of work from prokaryotes to eukaryotes. First, the mechanism of conditional, ppGpp-dependent fork arrest at the replication checkpoints of Bacillus subtilis will be analyzed to investigate if long range DNA-DNA interaction promoted by the replication termination protein (RTP) is the mechanism that converts a weak replication pause site to an efficient terminus. The preceding experiments will make use of fluorescent in situ hybridization, formaldehyde crosslinking and DNA microarrays as the principal experimental tools. Second, the mechanistic aspects of interaction between Tus and DnaB of Escherichia coli will be investigated by reverse 2-hybrid analysis to identify mutations in the helicase DnaB that allow the helicase to pass through the barrier of the replication terminus. These mutants and their suppressors in Tus will be used, along with wild type proteins to study Tus-DnaB interaction by EM and 3D image reconstruction. The main question that the principal investigator is trying to address is how does the contact between Tus and DnaB block the helicase activity of the latter and whether the physical contact inhibits the DNA-dependent ATPase activity of DnaB. A novel replication activation-based 2-hybrid system will be used to identify if the Ter-Tus complex or the dif-XerC/D complex interact with proteins located at or near the z ring to localize and hold the Ter at the cell center during chromosome replication and segregation. Finally, a multifaceted approach involving yeast genetics and molecular biology will be used to identify the proteins that interact with Fob 1 to arrest forks at yeast rDNA. The principal investigator will also try to identify the component of the yeast replication apparatus that interacts with the termination complex.