Visualizing the Bacterial Replisome at Single-Molecule Resolution

描述(由申请人提供)：基因组DNA的准确和有效复制对所有生物体都至关重要。这一过程中的错误可能会导致突变，并最终导致癌症。DNA复制的重要任务是由一个多蛋白质复合体--复制体执行的，该复制体在生命的所有领域都具有广泛保守的特征。复制体的中心成分是DNA聚合酶，它是一种在加工因子和其他辅助蛋白的帮助下合成DNA的酶。在真核生物中，染色体复制需要三种不同的聚合酶。这与模型细菌系统--大肠杆菌--形成了鲜明对比，后者只有一个复制聚合酶。最近，人们发现像枯草芽孢杆菌这样的低GC革兰氏阳性细菌需要两种复制聚合酶PolC和DNAE。关于这些聚合酶的作用和它们的活性协调机制的问题仍然存在。与Polc不同的是，Dna E缺乏校对结构域，容易出错；因此，在复制过程中适当调节Dna E的活性对细胞至关重要。对枯草杆菌如何协调聚合酶活性的详细分子水平的了解将为在更复杂的真核系统中如何调控复制聚合酶提供洞察力。这项研究将利用新颖的单分子方法来解决以下具体目标：目标1：确定枯草杆菌复制体的结构和组织原则。枯草杆菌中复制体的组织在关键方面与大肠杆菌中的复制体组织不同，这可能是因为需要协调两种不同的聚合酶。这一目标将利用体内荧光融合蛋白的单分子成像来确定重要复制体组分的拷贝数，如聚合酶PolC和DNAE、滑动夹子DNAN和夹子加载器复合体的τ亚单位。有助于组织复制体的蛋白质-蛋白质相互作用将通过对这些相互作用中涉及的结构域进行定向突变来识别。目的2：研究Polc和DNAE活动是如何在复制分叉上协调的。在目前关于Polc和Dna E作用的模型中，这两个聚合酶顺序地作用于滞后的链，这意味着在复制过程中必须重复进行聚合酶交换。这种聚合酶开关事件是如何调节的，以及与复制体组件的相互作用如何调节交换，目前尚不清楚。这些问题将通过利用单分子成像来测量枯草杆菌活细胞中复制叉处Polc和DNAE的寿命来解决。单个Polc和Dna E分子的动力学在整体生化实验中被掩盖，这将有助于阐明它们在复制过程中的作用。更有针对性的聚合酶交换机制研究将使用体外单分子DNA合成实验进行，该实验涉及最小重组的复制体。这项测试将揭示聚合酶交换的动力学，并将有助于确定这一过程中涉及的蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): The accurate and efficient replication of genomic DNA is critical for all organisms. Errors in this process can lead to mutations and ultimately to cancer. The important task of DNA replication is performed by a multi- protein complex, the replisome, which shares broadly conserved features across all domains of life. The central component of the replisome is DNA polymerase, the enzyme that synthesizes DNA with the help of processivity factors and other accessory proteins. In eukaryotes, three different polymerases are needed for chromosomal replication. This is in stark contrast to the model bacterial system, Escherichia coli, which has a single replicative polymerase. More recently, it has been discovered that two replicative polymerases, PolC and DnaE, are required in low-GC Gram-positive bacteria like Bacillus subtilis. Questions remain about the role of these polymerases and the mechanisms by which their activity is coordinated. Unlike PolC, DnaE lacks a proofreading domain and is error-prone; thus the proper regulation of DnaE activity during replication is critical for the cell. A detailed molecular-level understanding of how polymerase activity is coordinated by B. subtilis will provide insights into how replicative polymerases are regulated in more complex eukaryotic systems. This research will utilize novel single-molecule approaches to address the following specific aims: Aim 1: Determine the architecture and organizing principles of the B. subtilis replisome. It is becoming clear that the organization of the replisome in B. subtilis differs in key ways from that in E. coli, likely becaue of the need to coordinate two different polymerases. This aim will utilize in vivo single-molecule imaging of fluorescent fusion proteins to determine the copy number of important replisome components like the polymerases PolC and DnaE, the sliding clamp DnaN, and the τ subunit of the clamp loader complex. The protein-protein interactions that help to organize the replisome will be identified by making targeted mutations to domains implicated in these interactions. Aim 2: Investigate how PolC and DnaE activity is coordinated at the replication fork. In a current model for the role of PolC and DnaE, the two polymerases act sequentially on the lagging strand, meaning that polymerase exchange must occur repeatedly during replication. How such polymerase switching events are regulated and how interactions with replisome components mediate the exchange is unknown. These questions will be addressed by utilizing single-molecule imaging to measure the lifetimes of PolC and DnaE at the replication fork in live B. subtilis cells. The dynamics of individual PolC and DnaE molecules, which are obscured in ensemble biochemical experiments, will help to elucidate their roles during replication. A more targeted mechanistic investigation of polymerase exchange will be performed using an in vitro single- molecule DNA synthesis assay involving a minimally reconstituted replisome. This assay will reveal the kinetics of polymerase exchange and will help identify the protein-protein interactions involved in this process.