Mechanisms of Escherichia Coli Chromosomal Replication

描述(由申请人提供)：特定目的：DNAA蛋白通过策划一系列事件来调控大肠杆菌染色体DNA复制的启动，以在染色体起源ORIC组装复制分叉机制。一旦酶机器组装好，基因组就会随之复制。本研究的长期目标是了解大肠杆菌染色体DNA的复制过程及其在生化水平上的调控。我们分离了在启动过程中高度活跃的Dna A突变。在目标1中，他们的研究将揭示控制启动过程的调控机制。在目标2中，我们将描述DPS抑制DNAA功能的机制，DPS是一种在氧化应激、饥饿和细胞进入稳定期时表达的蛋白质。当大肠杆菌适应这些生长条件时，DPS可能会调节DNAA蛋白的活性，以调节启动过程。我们将在目标3中研究一个突变体Dna A，它在启动时存在缺陷，因为它显然无法感知ATP的水解。作为ATPase AAA家族中的一员，DNAA在ATP水解过程中可能会发生构象变化，从而调节其起始功能。DNAC也是AAA家族成员，并且是启动所必需的。我们将通过遗传和生化相结合的方法研究AIM 4中的DNAC功能，以了解它是如何将DNAB运送到ORIC的。最近对这个模型系统的研究表明，启动过程与真核细胞中发生的过程有惊人的相似之处。进一步研究在大肠杆菌中启动及其调控的分子机制，将有助于深入了解在其他生物中启动及其控制的生物化学。

DESCRIPTION (provided by applicant): Specific Aims: DnaA protein regulates the initiation of DNA replication of the Escherichia coli chromosome by orchestrating a series of events to assemble the replication fork machinery at oriC, the chromosomal origin. Once the enzymatic machinery has been assembled, duplication of the genome follows. The long term objective of this research is to understand the process of E. coli chromosomal DNA replication and its regulation at the biochemical level. We isolated dnaA mutations that are hyperactive in initiation. In Aim 1, their study will reveal regulatory mechanisms that control the initiation process. In Aim 2, we will characterize the mechanism of inhibition of DnaA function by Dps, a protein expressed in response to oxidative stress, starvation, and as cells enter stationary phase. Dps may modulate the activity of DnaA protein to regulate the initiation process as E. coli adapts to these growth conditions. We will study in Aim 3 a mutant DnaA that is defective in initiation because it apparently fails to sense the hydrolysis of ATP. As a member of the AAA+ family of ATPases, DnaA may undergo a conformational change upon ATP hydrolysis to regulate its function in initiation. DnaC is also an AAA+ family member, and is essential for initiation. We will study DnaC function in Aim 4 via combined genetic and biochemical approaches to understand how it delivers DnaB to oriC. Recent studies of this model system reveal that the initiation process bears striking similarities to that occurring in eukaryotic cells. Further study on the molecular mechanism of initiation and its regulation in E. coli should provide insight into the biochemistry of initiation and its control in other organisms.