Mechanisms of Escherichia Coli Chromosomal Replication

描述（由申请人提供）：具体目的：DnaA蛋白通过在染色体起源oriC上安排一系列事件组装复制叉机制来调节大肠杆菌染色体的DNA复制起始。一旦酶的机制被组装起来，基因组的复制就会随之而来。本研究的长期目标是了解大肠杆菌染色体DNA复制的过程及其在生化水平上的调控。我们分离出起始阶段异常活跃的dna突变。在Aim 1中，他们的研究将揭示控制启动过程的调节机制。在Aim 2中，我们将描述Dps抑制DnaA功能的机制，Dps是一种响应氧化应激、饥饿和细胞进入静止期时表达的蛋白质。当大肠杆菌适应这些生长条件时，Dps可能通过调节dna蛋白的活性来调节起始过程。我们将在Aim 3中研究一种突变dna，它在起始阶段有缺陷，因为它显然无法感知ATP的水解。作为AAA+ ATP酶家族的一员，dna在ATP水解过程中可能发生构象变化，调控其起始功能。DnaC也是AAA+家族成员，是入会必不可少的。我们将通过遗传和生化相结合的方法研究DnaC在Aim 4中的功能，以了解它如何将DnaB传递到oriC。最近对该模型系统的研究表明，起始过程与真核细胞中发生的过程有着惊人的相似性。进一步研究大肠杆菌起始的分子机制及其调控，为其他生物起始的生物化学及其控制提供依据。

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.