Mechanism of Termination of DNA Replication

DNA复制终止机制

• 批准号: 6438884
• 负责人: DEEPAK BASTIA
• 金额: $ 32.26万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6438884
• 关键词: Bacillus subtilis; DNA binding protein; DNA replication; DNA replication origin; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; cell cycle; chimeric proteins; fluorescence microscopy; fluorescent in situ hybridization; fungal genetics; gene expression; gene mutation; helicase; microorganism reproduction; nucleoproteins; protein purification; protein structure function; site directed mutagenesis; transcription termination; yeasts

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.

描述（由申请人提供）：本申请描述了实验 旨在解决有关机制的两个重要问题 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 酵母 rDNA 中的终止，因此代表工作从 原核生物到真核生物。一、机制有条件，ppGpp依赖 枯草芽孢杆菌复制检查点的分叉停滞将是 分析以研究长程 DNA-DNA 相互作用是否由 复制终止蛋白（RTP）是将弱复制蛋白转化为复制终止蛋白的机制。 复制暂停站点到高效终点。前面的实验将 利用荧光原位杂交、甲醛交联和 DNA微阵列作为主要的实验工具。二、机械方面 Tus 和大肠杆菌 DnaB 之间相互作用的方面将是 通过反向 2 杂交分析来鉴定解旋酶中的突变 DnaB 允许解旋酶穿过复制屏障 终点站。 Tus 中的这些突变体及其抑制子将与 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 地址是Tus和DnaB之间的联系如何阻止解旋酶 后者的活动以及身体接触是否抑制 DnaB 的 DNA 依赖性 ATP 酶活性。一种基于复制激活的新型 2-混合系统将用于识别 Ter-Tus 复合体或 diff-XerC/D 复合物与位于或靠近 z 环的蛋白质相互作用 在染色体复制过程中将 Ter 定位并保持在细胞中心 隔离。最后，涉及酵母遗传学和 分子生物学将用于识别与 Fob 相互作用的蛋白质 1 阻止酵母 rDNA 上的叉子。首席研究员还将尝试 识别与相互作用的酵母复制装置的组件 终止复合体。