Timing the bacterial replisome in live cells

生长中的细菌细胞与细胞分裂协调生长以维持细胞大小。这就要求相关的细胞过程在细胞诞生后的特定时间发生，并在规定的时间内完成。在细胞周期中，时间是如何在细胞中被追踪的仍然没有答案。在这里，我建议研究在DNA复制中发挥作用的蛋白质如何参与时间起始，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时间周期性如何在DNA复制中实现。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力学。目标1，在细胞周期中表征启动蛋白。DNA复制开始于DNA复制机制的装载。启动蛋白dna解开染色体的一个位点，并在细胞周期的特定时间帮助调解这一过程，但触发其活性的原因尚不清楚。我假设起始的时间是由细胞周期中溶液中dna的化学计量变化控制的。在这里，我们将描述扩散DNA的化学计量学，这是目前启动调节模型中忽略的一个方面，并描述其与DNA的结合。目的2，表征细胞中起始蛋白SeqA和Hda的动态。控制起始的蛋白质活性的时间并不局限于dna，而是延伸到它的一些调节因子，如SeqA和Hda。它们的活性通过直接与染色体结合或通过蛋白质的调解来调节。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：描述引物酶控制冈崎片段长度的过程。由于DNA的结构，一条链——滞后链——的合成不能连续进行。相反，它需要每几千个碱基对开始一次合成。复制体的一个亚基，称为引物酶，合成用于在滞后链上启动新的DNA片段的RNA引物。根据文献，我假设启动的时间是由启动酶被招募到复制体的频率决定的。在这里，我打算描述引物酶的动力学及其与复制机制的协调。这项工作的结果将有助于我们了解细菌中DNA复制及其与细胞周期的关系。我希望我们开发的显微镜技术、图像分析工具和遗传系统将对其他研究人员有所帮助。此外，该项目的跨学科性质将为高素质的个人提供广泛的技能，以发展学术界或工业界的职业生涯。从长远来看，这一提议将有助于使我的实验室在DNA复制起始研究方面处于领先地位。

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.