Regulation of initiation of replication in bacteria

细菌复制起始的调节

• 批准号: 141866389
• 负责人: Professor Dr. Peter Graumann
• 金额: --
• 依托单位: Zentrum für Synthetische Mikrobiologie (SYNMIKRO)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/141866389?language=en
• 关键词: Regulation; initiation; replication; bacteria

All cells must ensure that initiation of replication occurs only once per cell cycle, and that it is tightly linked to the physiological state of the cell. Alterations in initiation control lead to slow growth, cell death and to carcinogenesis. In all bacteria investigated so far, the highly conserved protein DnaA controls initiation of replication. DnaA is a sequence specific DNA-binding ATPase that is homologous to eukaryotic Orc proteins, which are also crucial for initiation control. Understanding of what triggers DnaA to unwind origin regions and load the replication machinery is a key question in biology, yet unresolved. We have recently been able to visualize GFP-DnaA and to follow its binding to replication origins and to the replisome in live cells. We have found a novel mechanism for replication control: after initiation of replication, DnaA is tethered to the replisome via YabA, and is spatially sequestered from the origins that are moved away from the replisome, thus preventing reinitiation. We will now address the question of what regulates DnaA activity at origin regions: a) analysis of the localization and effect of mutant forms of GFP-DnaA, b) effect of a novel regulator, Soj, on DnaA in vivo and in vitro, c) the identification of novel DnaA binding partners, and d) a suppressor screen for the bypass of a dominant negative dnaA allele.

所有细胞必须确保每个细胞周期只启动一次复制，并且复制与细胞的生理状态紧密相连。起始控制的改变会导致生长缓慢、细胞死亡和致癌。到目前为止，在所有被研究的细菌中，高度保守的蛋白质Dna A控制着复制的启动。DNAA是一种序列特异性的DNA结合ATPase，与真核ORC蛋白同源，ORC蛋白也是启动控制的关键。了解是什么触发DNAA解开起始区并加载复制机制是生物学中的一个关键问题，但尚未解决。最近，我们已经能够可视化GFP-DNAA，并跟踪其与复制起点和活细胞中的复制体的结合。我们发现了一种新的复制控制机制：复制开始后，Dna A通过yaba被拴在复制体上，并在空间上与远离复制体的起始点隔离，从而防止再次启动。我们现在将讨论在起源区调节DNAA活性的问题：a)GFP-DNAA突变形式的定位和影响的分析，b)新型调节剂SOJ在体内和体外对DNAA的影响，c)新的DNAA结合伙伴的鉴定，以及d)显性负DNAA等位基因旁路的抑制筛选。