Cell Cycle Assembly of Nucleoprotein Complexes

核蛋白复合物的细胞周期组装

• 批准号: 6779513
• 负责人: ALAN Carl LEONARD
• 金额: $ 22.48万
• 依托单位: FLORIDA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779513
• 关键词: DNA binding protein; DNA footprinting; DNA replication origin; Escherichia coli; bacterial genetics; binding sites; cell cycle; cell growth regulation; flow cytometry; gene mutation; microorganism culture; microorganism growth; nucleoproteins; site directed mutagenesis

DESCRIPTION (provided by applicant): This application focuses on DNA-protein interactions that precisely time new rounds of chromosome replication, with the long-term objective of dissecting molecular mechanisms controlling bacterial growth. E. coli chromosomal DNA replication is triggered during each cell cycle by pre-replication complexes (pre-RC) that unwind origin (oriC) DNA. In normal pre-RC assembly, initiator DnaA interacts with at least eight binding sites at the correct cell cycle times. Our major goal is to understand how intrinsic nucleotide sequence of oriC determines temporal regulation of ordered assembly, the functional requirement for ATP-DnaA, synchronous firing of multiple origins, and one initiation per cycle control. Key experiments focus on newly discovered DnaA binding sites (I2 and I3), which are required for ATP-DnaA dependent unwinding, play a role in Fis and IHF regulation of pre-RC assembly, and contain the deoxyadenosine methyltransferase recognition sequence, GATC. The Specific Aims are as follows: 1. To use mutagenesis, DNA footprinting, and unwinding assays to test the hypotheses that DnaA binding sites are critically positioned in oriC and ordered DnaA binding is required for correct pre-RC assembly; 2. To evaluate ordered DnaA loading and the interplay between DnaA, Fis and IHF binding at oriC sites as determinants of initiation timing and synchrony using age-selected E. coli and flow cytometry; 3. To use mutagenesis, DNA footprinting and chemical assays to test the hypothesis that DnaA recognition sites 12, 13 and the IHF binding site are responsive to DNA methylation state and/or sequestration; and 4. To test the hypothesis, using mutagenesis and chemical assays, that DnaA binding sites within the A-T rich, 13-mer unwinding region regulate the location of strand separation and repress unwinding at high initiator levels. Our continued dissection of the triggering mechanism for chromosomal DNA synthesis in E. coli should provide new insight into the function of growth regulatory machinery in all living cells, particularly cell cycle-specific switches. Identification of new features of the pre-RC is also immensely important for understanding the control of bacterial growth, as well as cell growth defects, and can help to identify new targets used to guide the design of novel cell growth inhibitors.

描述（由申请人提供）：本申请专注于DNA-蛋白质相互作用，其精确地确定新一轮染色体复制的时间，长期目标是剖析控制细菌生长的分子机制。E.大肠杆菌染色体DNA复制在每个细胞周期期间由解绕起始（oriC）DNA的复制前复合物（pre-RC）触发。在正常的前RC组装中，起始DnaA在正确的细胞周期时间与至少八个结合位点相互作用。我们的主要目标是了解oriC的内在核苷酸序列如何决定有序组装的时间调节，ATP-DnaA的功能要求，多个起源的同步发射，以及每个周期控制一次启动。关键实验集中在新发现的DnaA结合位点（I2和I3），这是所需的ATP-DnaA依赖解旋，发挥作用的Fis和IHF调节前RC组装，并包含脱氧腺苷甲基转移酶识别序列，GATC。具体目标如下：1.使用突变、DNA足迹和解旋测定来测试DnaA结合位点在oriC中的关键位置以及正确的预RC组装需要有序的DnaA结合的假设; 2.利用年龄选择的E.大肠杆菌和流式细胞术; 3.使用诱变、DNA足迹和化学测定来测试DnaA识别位点12、13和IHF结合位点响应于DNA甲基化状态和/或隔离的假设;和4.为了验证这一假设，使用诱变和化学测定，即富含A-T的13-mer解旋区域内的DnaA结合位点调节链分离的位置并在高起始水平下抑制解旋。我们继续剖析了大肠杆菌染色体DNA合成的触发机制。大肠杆菌应该提供新的洞察力的功能，生长调节机制在所有活细胞，特别是细胞周期特异性开关。鉴定前RC的新特征对于理解细菌生长的控制以及细胞生长缺陷也非常重要，并且可以帮助鉴定用于指导新型细胞生长抑制剂设计的新靶标。