Mechanism of Termination of DNA Replication

描述（由申请人提供）：本申请描述了实验 旨在解决两个重要问题的机制， 原核生物中的复制叉停滞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 终止在酵母rDNA，因此代表了工作的过渡， 原核生物到真核生物。第一，有条件的机制，ppGpp依赖 在枯草芽孢杆菌的复制检查点处的叉停滞将是 分析以研究是否由DNA促进的长距离DNA-DNA相互作用。 复制终止蛋白（RTP）是将弱的 复制暂停位点到有效的末端。之前的实验将 利用荧光原位杂交，甲醛交联， DNA微阵列作为主要的实验工具。第二，机械化 Tus和DnaB之间的相互作用方面的大肠杆菌将是 通过反向双杂交分析研究以鉴定解旋酶中的突变 允许解旋酶通过复制屏障的DnaB 终点站这些突变体和它们的抑制子将在Tus中使用，沿着 通过EM和3D图像研究Tus-DnaB相互作用的野生型蛋白 重建首席研究员试图解决的主要问题是， 地址是Tus和DnaB之间的接触如何阻止解旋酶 后者的活动以及身体接触是否抑制了 DnaB的DNA依赖性ATP酶活性。一种基于复制激活的 2-混合系统将被用来确定是否Ter-Tus复杂或 dif-XerC/D复合物与位于z环或z环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号引物在酵母rDNA上捕获叉。首席研究员还将尝试 鉴定酵母复制装置的与以下物质相互作用的组分： 终止复合体。

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.