Regulation of initiation of replication in bacteria

所有细胞都必须确保每个细胞周期只发生一次开始复制，并且它与细胞的生理状态密切相关。起始控制的改变导致生长缓慢、细胞死亡和致癌。在目前研究的所有细菌中，高度保守的蛋白质dna控制着复制的起始。dna是一种序列特异性dna结合atp酶，与真核Orc蛋白同源，对起始控制也至关重要。了解是什么触发dna解开起始区域并加载复制机制是生物学中的一个关键问题，但尚未解决。我们最近已经能够可视化gfp - dna，并跟踪其与活细胞中的复制起源和复制体的结合。我们发现了一种新的复制控制机制：在复制开始后，dna通过YabA连接到复制体上，并在空间上与从复制体移开的起点隔离，从而阻止了复制的重新启动。我们现在将讨论是什么调控了起源区域的dna活性：a)分析GFP-DnaA突变形式的定位和影响，b)一种新的调节剂Soj对体内和体外dna的影响，c)鉴定新的dna结合伙伴，d)抑制筛选，以绕过显性阴性dna等位基因。

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.