Mechanism of Termination of DNA Replication

DNA复制终止机制

• 批准号: 6622077
• 负责人: DEEPAK BASTIA
• 金额: $ 34.32万
• 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/6622077
• 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）：此申请描述实验