Timing the bacterial replisome in live cells

活细胞中细菌复制体的计时

• 批准号: RGPIN-2019-05701
• 负责人: ReyesLamothe, Rodrigo
• 金额: $ 2.33万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745865
• 关键词: Timing; bacterial; replisome; live; cells

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.

生长的细菌细胞协调生长和细胞分裂以维持细胞大小。这要求相关的细胞过程在细胞出生后的特定时间发生，并且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，我建议研究蛋白质如何在DNA复制参与定时启动，并在DNA合成过程中的事件。我研究计划的总体目标是了解DNA复制中如何实现时间周期性。我们将使用活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、研究启动子蛋白在细胞周期中的作用。DNA复制开始于DNA上的复制机器的加载。起始蛋白DnaA解开染色体的一个位点，并在细胞周期的特定时间帮助介导这一过程，但触发其活性的原因尚不清楚。我推测，启动的时间是由在细胞周期的溶液中的DnaA的化学计量的变化控制。在这里，我们将描述扩散DnaA的化学计量，在目前的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、研究起始蛋白SeqA和Hda在细胞内的动态变化。蛋白质活性控制起始的时间并不限于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直接结合或通过蛋白质介导来调节。因此，我假设细胞控制它们与染色体结合的时间。目的3，描述引发酶对冈崎片段长度的调控过程。由于DNA的结构，其中一条链的合成-滞后链-不能连续进行。相反，它需要每隔几个酶对就开始合成。复制体的一个亚基，称为引物酶，合成RNA引物，用于启动滞后链的新DNA片段。根据文献，我假设启动的时间是由启动酶被募集到复制体的频率决定的。在这里，我计划的特点，引发酶的动态和它的协调与复制机制。这项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镜技术、图像分析工具和遗传系统能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，这个建议将有助于建立我的实验室作为一个领导者在研究DNA复制的启动。