Cell Cycle Assembly of Nucleoprotein Complexes

描述（由申请人提供）：该申请专注于dna -蛋白质相互作用，精确定时新一轮染色体复制，长期目标是解剖控制细菌生长的分子机制。大肠杆菌染色体DNA复制在每个细胞周期中由预复制复合体（pre-RC）触发，该复合体解除起源（oriC） DNA。在正常的pre-RC组装中，启动dna在正确的细胞周期时间内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 - dna的功能要求，多个起源的同步发射，以及每个周期控制的一个起始。关键实验集中在新发现的dna结合位点（I2和I3），这些位点是ATP-DnaA依赖性解绕所必需的，在Fis和IHF调控pre-RC组装中起作用，并且包含脱氧腺苷甲基转移酶识别序列GATC。具体目标如下：1。使用诱变、DNA足迹和解绕分析来验证DNA结合位点在oriC中的关键位置和正确的pre-RC组装所需的有序DNA结合的假设；2. 使用年龄选择的大肠杆菌和流式细胞术评估有序的dna负载以及dna、Fis和IHF在oriC位点结合之间的相互作用，作为起始时间和同步的决定因素；3. 利用诱变、DNA足迹和化学分析来验证DNA识别位点12、13和IHF结合位点对DNA甲基化状态和/或封存有反应的假设；和4。为了验证这一假设，使用诱变和化学分析，在富含A-T的13-mer解绕区域内的dna结合位点调节链分离的位置，并在高引发剂水平上抑制解绕。我们对大肠杆菌染色体DNA合成触发机制的持续剖析，将为所有活细胞，特别是细胞周期特异性开关的生长调节机制的功能提供新的见解。识别pre-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，并且可以帮助识别用于指导设计新型细胞生长抑制剂的新靶点。

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.