Timing the bacterial replisome in live cells

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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.