Regulation of initiation of replication in bacteria

所有的细胞都必须确保复制的启动在每个细胞周期只发生一次，并且它与细胞的生理状态紧密相连。起始控制的改变导致生长缓慢、细胞死亡和致癌。在迄今为止研究的所有细菌中，高度保守的蛋白质DnaA控制复制的起始。DNA A是一种序列特异性的DNA结合ATP酶，与真核生物的Orc蛋白同源，这也是启动控制的关键。了解是什么触发DnaA解开起源区域并加载复制机器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-DnaA，并遵循其结合到复制起点和活细胞中的复制体。我们已经发现了一种新的复制控制机制：复制开始后，DnaA通过YabA与复制体相连，并在空间上与远离复制体的起点隔离，从而防止重新启动。我们现在将讨论是什么调节DnaA在起源区域的活性的问题：a）分析GFP-DnaA的突变形式的定位和作用，B）新型调节剂Soj在体内和体外对DnaA的作用，c）鉴定新型DnaA结合配偶体，和d）抑制剂筛选显性负性dnaA等位基因的旁路。

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.