TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

大肠杆菌中 DNA 复制的终止

• 批准号: 2181866
• 负责人: THOMAS M. HILL
• 金额: $ 7.47万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181866
• 关键词: DNA binding protein; DNA replication; Escherichia coli; bacterial genetics; crosslink; gene induction /repression; gene mutation; genetic terminator element; nucleic acid sequence; plasmids; polymerase chain reaction; protein structure; transcription termination; transfection

In the terminus region of the Escherichia coli chromosome, DNA replication forks are arrested at specific DNA sequences, called Ter sites. Arrest of DNA replication is mediated by the Tus protein, which binds to the Ter sites with high affinity. Binding of Tus to a Ter site forms an asymmetric protein-DNA complex that shows polarity of function; that is, it halts replication forks from one direction, but not the other. Thus, the Tus-Ter complex forms an orientation-dependent barrier to the progression of DNA replication. In the six years that have passed since the identification of Tus and the Ter sites, significant advances have been made characterizing the interactions between Tus and its cognate binding sequence and between Tus and the replisome during replication arrest. Nonetheless, the molecular mechanism of Tus-mediated arrest of DNA replication remains unknown. Likewise, our knowledge of the domains of the Tus protein that participate in DNA-binding or the DNA replication arrest function is largely incomplete. The experiments outlined in this proposal are directed towards a greater understanding of structure-function relationships in the Tus protein and the mechanism of replication arrest. We propose three specific goals: 1) Isolate extragenic suppressors of replication arrest using standard genetic techniques to identify gene products that participate in the replication arrest process. 2) Identify an in vitro system that accurately measures Tus function and utilize this system to examine basic questions about the process and mechanism of replication arrest. 3) Investigate the functional domains of the Tus protein using the following approaches: (a) cross-linking of Tus to a Ter site, (b) crystallizing the protein either alone or as a protein-DNA complex, and (c) linker scanner mutagenesis of individual domains of the protein.

在大肠杆菌染色体的末端区域，DNA