Timing the bacterial replisome in live cells

生长中的细菌细胞协调生长和细胞分裂，以保持细胞大小。这要求相关的细胞过程在细胞出生后的特定时间发生，并在规定的时间内完成。细胞周期中的时间是如何被跟踪的，目前还没有答案。在这里，我建议研究作用于DNA复制的蛋白质如何参与计时启动，以及DNA合成过程中的事件。我的研究计划的总体目标是了解DNA复制中时间周期性是如何实现的。我们将使用活的单细胞单分子显微镜来研究这些事件，以表征细胞中蛋白质的动态。 目的1，研究启动蛋白在细胞周期中的特性。DNA复制始于将复制机制加载到DNA上。启动子蛋白Dna A解开染色体的一个部位，并在细胞周期的特定时间帮助调节这一过程，但什么触发了它的活动尚不清楚。我假设，启动的时间是由细胞周期中溶液中DNAA的化学计量比的变化控制的。在这里，我们将表征扩散DNAA的化学计量学，这是当前启动调节模型中忽略的一个方面，并表征其与DNA的结合。 目的2，研究起始蛋白SEQA和HDA在细胞内的动态变化。控制起始的蛋白质活性的时间并不局限于DNAA，而是延伸到它的一些调节因子，如SEQA和HDA。它们的活性通过与染色体的结合来调节，直接或通过蛋白质的调节。因此，我假设细胞控制着它们与染色体结合的时间。在这里，我计划描述这些蛋白质的结合动力学和控制它的因素。 目的3、描述了Primase控制Okazaki片段长度的过程。由于DNA的结构，其中一条链的合成不能连续进行。相反，它需要每隔几个千碱基对启动合成。复制体的一个亚单位被称为启动酶，它合成用于在滞后链上启动新DNA片段的RNA引物。在文献的基础上，我假设启动的时机是由复制体招募启动酶的频率确定的。在这里，我计划描述Primase的动态以及它与复制机制的协调。 拟议工作的结果将有助于我们了解DNA复制及其与细菌细胞周期的关系。我希望我们开发的显微技术、图像分析工具和基因系统将对其他研究人员有所帮助。此外，该项目的跨学科特性将为高素质的个人提供广泛的技能，以便在学术界或行业发展事业。从长远来看，这项提议将有助于将我的实验室确立为DNA复制启动研究的领先者。

Growing bacterial cells coordinate growth with cell division to maintain cell size. This requires that relevant cellular processes occur at specific times after cell birth, and that they are accomplished within defined periods. How is time tracked in cells during the cell cycle is still unanswered. Here I propose to study how proteins acting in DNA replication participate in timing initiation, and events during DNA synthesis. The overall objective of my research program is to understand how time periodicity is achieved in DNA replication. We will study these events using live single-cell single-molecule microscopy to characterize the dynamics of proteins in cells. Aim 1, to characterise the initiator protein over the cell cycle. DNA replication starts with the loading of the replication machinery on DNA. The initiator protein, DnaA, unwinds a site of the chromosome and helps to mediate this process at a specific time of the cell cycle, but what triggers its activity is unknown. I hypothesize that timing of initiation is controlled by changes in the stoichiometry of DnaA in solution over the cell cycle. Here we will characterize the stoichiometry of diffusing DnaA, an aspect ignored in current models for initiation regulation, and characterize its binding to DNA. Aim 2, to characterise the dynamics of initiation proteins SeqA and Hda in cells. Timing of protein activity to control initiation is not restricted to DnaA, but extends to some of its regulators like SeqA and Hda. Their activity is regulated by binding to the chromosome, directly or by mediation of a protein. Hence, I hypothesize that the cell controls the timing of their binding to the chromosome. Here I plan to characterize the binding dynamics of these proteins and the factors that control it. Aim 3, to describe the process of Okazaki fragment length-control by primase. Due to the structure of DNA, synthesis of one the strands the lagging strand cannot be done continuously. Instead it requires the initiation of synthesis every few kilobase pairs. A subunit of the replisome, called the primase, synthesizes RNA primers used to initiate a new DNA fragment at the lagging strand. Based on the literature, I hypothesize that timing of priming is established by the frequency at which primase is recruited to the replisome. Here I plan to characterize the dynamics of primase and its coordination with the replication machinery. The outcome of the proposed work will help us understand DNA replication and its relationship with the cell cycle in bacteria. I expect that the microscopy techniques, image analysis tools and genetic systems that we develop will be helpful to other researchers. In addition, the interdisciplinary character of this projects will provide a wide set of skills for highly qualified personal to develop a career in academia or industry. In the long run, this proposal will help to establish my lab as a leader in the study of the initiation of DNA replication.