DNA Replication, Bacteria Cell Cycle, and Cell Growth

这个项目的目标是了解DNA复制的启动是如何与生长速度相耦合的。为了了解启动事件的频率是如何确定的，必须了解调节Dna A蛋白表达的机制。遗传学和生物化学都被用来阐明控制DNAA操纵子启动子表达的调控电路。生长速度调节剂和丙氨酸鸟苷四磷酸(PpGpp)抑制Dna A P2启动子的转录。这种抑制的分子基础正在体外表征。对ppGpp的抑制反应有缺陷的突变株将被检查生长速度调节和细胞周期中DNA复制的准确启动时间。我们将确定在缺乏ppGpp的细胞中，Dna A基因是否仍处于生长速度控制之下--除了ppGpp之外，是否有其他生长速度调节机制来控制Dna A蛋白的表达？我们将通过分离和鉴定具有改变的DNA结合特异性的DNAA突变蛋白来研究DNAA蛋白与DNA的相互作用。我们将构建一个在ORIC两侧缺失基因的染色体突变，并将检查所产生的突变是否在细胞周期中丢失了精确的计时。这项研究研究了细胞分裂中的一个关键步骤的控制，并将为生物学中的一个基本问题以及不受控制的细胞增殖等恶劣条件提供重要的见解。

The goal of this project is to understand how initiation of DNA replication is coupled to growth rate. To understand how the frequency of the initiation event is determined, the mechanisms that regulate the expression of the DnaA protein must be understood. Both genetics and biochemistry are being used to elucidate the regulatory circuits controlling expression of the dnaA operon promoters. The growth rate regulator and alarmone, guanosine tetraphosphate (ppGpp), inhibits transcription from the dnaA P2 promoter. The molecular basis of this inhibition is being characterized in vitro. Mutants defective in the inhibitory response to ppGpp will be examined for growth rate regulation and precise timing of initiation of DNA replication in the cell cycle. We will determine whether the dnaA gene is still under growth rate control in cells that lack ppGpp - is there a growth rate regulation mechanism other than ppGpp that controls the expression of DnaA protein? We will investigate DnaA protein-DNA interactions by isolation and characterization of DnaA mutant proteins with altered DNA-binding specificities. We will construct a chromosomal mutation with genes on either side of oriC deleted, and the resulting mutant will be examined for loss of precise timing in the cell cycle. %%% This research studies the control of a key step in cell divison and should provide significant insights into a basic question in biology, as well as abberant conditions suchs as uncontrolled cell mutiplication.