INITIATION OF DNA REPLICATION--DNAA, RNA POLYMERASE, & COUPLING TO CELL CYCLE

我们研究的长期目标是了解这些机制 调控DNA复制的启动。在原核生物和 真核细胞中，染色体复制的速度是由 复制起始点的启动速度，而不是链的速度 伸长率。因此，复制率的决定因素及其 控制是启动步骤中必须涉及的。在细菌中 细胞，在细菌起源处启动复制，ORIC，需要一个 RNA聚合酶介导的转录事件与DNAA的存在 蛋白。这个项目试图用分子术语来定义这些事件。我们 正在使用遗传和生物化学方法来阐明 RNA聚合酶参与启动，无论是启动还是 转录激活及DNAA蛋白与ORIC的相互作用 在形成最初的复合体时。只有对这些有详细的了解 对DNA复制的调控可以理解两个事件。此外, 我们计划研究DNAA基因的表达调控，因为 该基因的表达可能在将启动事件与 细胞周期。具体来说，我们计划： 。刻画与ORIC相邻的序列的参与 启动DNA复制。删除ORIC旁边的区域将 被引入到染色体中并进行特征分析。这些删除 将被转录终止序列和G C取代 街区。 。确定原始组份II体外系统是否需要RNA 聚合酶。 。定义结构特征和其他参数 形成了奥里克初始复合体。DNAA蛋白是否会引起一次弯曲？ DNAA结合部位和更多结合时对弯曲的贡献 按原样以平行位置或交替位置添加站点 在奥里克身上见过吗？ 。研究DNAA蛋白表达的调控。用我们的单轮 体外转录实验和定点突变技术用于评估 生长速度调节和其他控制的顺序规范 分子。寻找其他调节DNAA基因表达的因素 用凝胶移位分析和西南地区。

The long term goal of our research is to understand the mechanisms regulating the initiation of DNA replication. In both prokaryotic and eukaryotic cells, the rate of chromosomal duplication is controlled by the rate of initiation at the origin of replication, not by the rate of chain elongation. Consequently, the determinants of the replication rate and its control are necessarily involved in the initiation step. In the bacterial cell, initiation of replication at the bacterial origin, oriC, requires an RNA polymerase-mediated transcription event and the presence of the DnaA protein. This project seeks to define these events in molecular terms. We are using genetic and biochemical approaches to elucidate both the involvement of RNA polymerase in initiation, whether it be priming or transcriptional activation, and the interaction of DnaA protein with oriC in forming the initial complex. Only with a detailed knowledge of these two events can regulation of DNA replication be understood. In addition, we plan to study the regulation of expression of the dnaA gene, because expression of this gene may be critical in linking the initiation event to the cell cycle. Specifically, we plan to: . Characterize the involvement of sequences adjacent to oriC in initiation of DNA replication. Deletions of regions next to oriC will be introduced into the chromosome and characterized. These deletions will be replaced with transcription termination sequences and G+C blocks. . Establish if the original Fraction II in vitro system requires RNA polymerase. . Define the structural features and other parameters that contribute to forming the oriC initial complex. Does DnaA protein induce bending at one DnaA binding site and what contributions to bending occur when more binding sites are added, in parallel positions or in alternating positions, as is seen in oriC? . Investigate regulation of DnaA protein expression. Use our single round in vitro transcription assay and site-directed mutagenesis to assess sequence specifications for growth rate regulation and other controlling molecules. Search for other factors that regulate dnaA gene expression with gel shift assays and Southwesterns.