Mechanisms of Escherichia Coli Chromosomal Replication

描述（由申请方提供）：特定目的：DnaA蛋白通过协调一系列事件在oriC（染色体起点）组装复制叉机制来调节大肠杆菌染色体DNA复制的起始。一旦酶机制组装完成，基因组的复制就随之而来。本研究的长期目标是了解E.大肠杆菌染色体DNA复制及其在生化水平上的调控。 我们分离出了在启动中异常活跃的dnaA突变。在目标1中，他们的研究将揭示控制启动过程的调节机制。在目标2中，我们将表征Dps抑制DnaA功能的机制，Dps是一种响应氧化应激、饥饿和细胞进入稳定期而表达的蛋白质。Dps可能通过调节DnaA蛋白的活性来调节启动过程，E.大肠杆菌适应这些生长条件。我们将在目标3中研究一种突变的DNA A，它在起始时有缺陷，因为它显然不能感知ATP的水解。作为ATP酶AAA+家族的成员，DnaA可在ATP水解时经历构象变化以调节其起始功能。DnaC也是AAA+家族成员，对启动至关重要。我们将通过结合遗传和生物化学方法研究Aim 4中的DnaC功能，以了解它如何将DnaB传递给oriC。最近对该模型系统的研究表明，启动过程与真核细胞中发生的过程具有惊人的相似性。进一步研究了E.对大肠杆菌的研究将为了解其他生物的启动和控制的生物化学提供帮助。

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.