Cell Cycle Assembly of Nucleoprotein Complexes

描述(由申请人提供)：这项申请专注于DNA-蛋白质相互作用，准确地计算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。大肠杆菌染色体DNA复制是在每个细胞周期中由复制前复合体(Pre-RC)触发的，它解开起源(ORIC)的DNA。在正常的Pre-RC组装中，启动子Dna A在正确的细胞周期时间至少与8个结合位点相互作用。我们的主要目标是了解ORIC的固有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、多个起始点的同步启动以及每个周期一次启动控制。关键实验集中在新发现的DNAA结合位点(I2和I3)，它们是ATP-DNAA依赖解离所必需的，在FIS和IHF调节前RC组装中发挥作用，并包含脱氧腺苷甲基转移酶识别序列GATC。其具体目的如下：1.使用突变、DNA足迹和解离分析来验证DNAA结合位点位于ORIC中的关键位置以及正确的前RC组装所需的有序DNAA结合的假设；2.使用年龄选择的大肠杆菌和流式细胞术来评估有序的DNAA装载以及ORIC位点上DNAA、FIS和IHF结合作为起始时机和同步性的决定因素；3.使用突变、DNA足迹和化学分析来检验DNAA识别位点12、13和IHF结合位点对DNA甲基化状态和/或隔离的响应的假设；4.为了验证这一假设，使用突变和化学分析方法，即富含A-T的13-聚体解旋区内的DNAA结合位点调节高启动子水平的链分离和抑制解离的位置。我们继续剖析大肠杆菌中染色体DNA合成的触发机制，应该会为所有活细胞中生长调节机制的功能提供新的见解，特别是细胞周期特异性开关。识别前RC的新功能对于理解细菌生长的控制以及细胞生长缺陷也是极其重要的，并有助于识别用于指导新型细胞生长抑制物设计的新靶点。

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.