Assembly of the bacterial DNA replication initiation complex

细菌 DNA 复制起始复合物的组装

• 批准号: BB/K017527/1
• 负责人: Heath Murray
• 金额: $ 38.66万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK017527%2F1
• 关键词: Assembly; bacterial; DNA; replication; initiation

The cell is the basic unit of structure for all living organisms. For a cell to grow and divide it must follow a blueprint that provides the instructions describing how to perform these essential activities. In all cells this information is encoded within DNA. Every time a cell divides it must replicate its DNA and pass on one complete, undamaged copy to each progeny cell. DNA replication has to be tightly controlled to ensure that each newborn cell contains the correct amount of genetic information. If DNA replication is delayed, then upon cell division one daughter cell will fail to inherit a full complement of the genetic information and will be inviable. If DNA replication occurs earlier than needed the cell will contain too many copies of its genes, leading to altered levels of expression which can cause developmental defects.The start of DNA replication requires dedicated replication initiator proteins. Initiator proteins bind to sites termed origins of replication where they act to recruit the DNA replication machinery. Throughout the three kingdoms of life, all initiator proteins contain a related protein fold (the initiator specific AAA+ motif), suggesting that they share common activities required for their activity. Bacteria, with their relatively simple and well characterised structure and physiology, are ideal systems with which to study the molecular mechanisms of DNA replication because they are readily amenable to genetic manipulation and their proteins tend to be tractable subjects for biochemical and structural analyses. The bacterial DNA replication machinery is also an attractive target for potential antibiotics because it is essential for growth and it is currently free from problems of pre-existing resistance.The bacterial DNA replication initiator protein is called DnaA. DnaA binds to specific sequences within the bacterial replication origin and forms a large nucleoprotein complex that separates the two strands of the DNA duplex to initiate the process of genome duplication. Excitingly, a molecular basis for replication origin opening by DnaA is beginning to emerge. Structural studies using x-ray crystallography have found that DnaA assembles into a helical filament that stretches DNA to promote opening of the replication origin. While these structure-based studies are imperative to derive a molecular understanding of DnaA activity, it is important to note that they are limited because they only provide a static image of the dynamic DNA replication initiation reaction. Furthermore, DnaA was not crystallized in the presence of replication origin DNA. Therefore, the structures of DnaA do not reveal how the protein initially assembles into an oligomer at the replication origin, nor do they reveal how DnaA transitions into the conformation that is thought to stretch and open DNA. Previous work in my laboratory has established a novel biochemical assay using purified proteins that detects DnaA helix formation. We have recently improved this methodology and are now able to demonstrate that DnaA adopts at least two distinct helical assembly states specifically at the replication origin.The purpose of this research project is to investigate the pathway of DNA replication initiation by identifying the sequences within the replication origin that are required for assembling the initial DnaA helix and the sequences that are required for promoting the transition between different DnaA conformations. We will utilize genetic approaches to dissect the origin region in vitro and in vivo and then we will utilize our novel helix formation assay to determine how these changes affect DnaA. This project will provide cutting-edge knowledge and will underpin future studies regarding a fundamental biological question that is essential for cellular viability and proliferation.

细胞是所有生物体的基本结构单位。为了让细胞生长和分裂，它必须遵循一个蓝图，该蓝图提供了描述如何执行这些基本活动的说明。在所有细胞中，该信息都编码在 DNA 中。细胞每次分裂时都必须复制其 DNA，并将一份完整的、未损坏的副本传递给每个子代细胞。 DNA 复制必须受到严格控制，以确保每个新生细胞包含正确数量的遗传信息。如果DNA复制被延迟，那么在细胞分裂时，一个子细胞将无法继承全部的遗传信息，并且将无法生存。如果 DNA 复制发生得早于所需时间，细胞将包含过多的基因拷贝，导致表达水平改变，从而导致发育缺陷。DNA 复制的开始需要专用的复制起始蛋白。起始蛋白与被称为复制起点的位点结合，在那里它们发挥作用来招募 DNA 复制机制。在生命的三个王国中，所有起始蛋白都包含相关的蛋白折叠（起始蛋白特异性 AAA+ 基序），这表明它们具有其活性所需的共同活性。细菌具有相对简单且特征明确的结构和生理学，是研究 DNA 复制分子机制的理想系统，因为它们很容易进行基因操作，并且它们的蛋白质往往是生化和结构分析的易处理对象。细菌 DNA 复制机制也是潜在抗生素的一个有吸引力的目标，因为它对于生长至关重要，而且目前不存在预先存在的耐药性问题。细菌 DNA 复制起始蛋白称为 DnaA。 DnaA 与细菌复制起点内的特定序列结合，形成大型核蛋白复合物，将 DNA 双链体的两条链分开，从而启动基因组复制过程。令人兴奋的是，DnaA 打开复制起点的分子基础开始出现。使用 X 射线晶体学进行的结构研究发现，DnaA 组装成螺旋丝，拉伸 DNA 以促进复制起点的打开。虽然这些基于结构的研究对于从分子角度理解 DnaA 活性至关重要，但值得注意的是它们的局限性，因为它们仅提供动态 DNA 复制起始反应的静态图像。此外，在复制起点DNA存在的情况下，DnaA不会结晶。因此，DnaA 的结构并没有揭示蛋白质最初如何在复制起点组装成寡聚物，也没有揭示 DnaA 如何转变为被认为可以拉伸和打开 DNA 的构象。我实验室之前的工作已经建立了一种使用纯化蛋白质检测 DnaA 螺旋形成的新型生化测定方法。我们最近改进了这种方法，现在能够证明 DnaA 在复制起点处采用至少两种不同的螺旋组装状态。该研究项目的目的是通过识别复制中的序列来研究 DNA 复制起始的途径组装初始 DnaA 螺旋所需的起点和促进不同 DnaA 构象之间转变所需的序列。我们将利用遗传方法在体外和体内剖析起源区域，然后利用我们的新型螺旋形成测定来确定这些变化如何影响 DnaA。该项目将提供前沿知识，并将为未来有关细胞活力和增殖至关重要的基本生物学问题的研究奠定基础。

项目成果

Probing Chromosome Dynamics in Bacillus subtilis.

探索枯草芽孢杆菌的染色体动力学。

• DOI: http://dx.10.1007/978-1-4939-3631-1_8
• 发表时间: 2016
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Koh A

Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

枯草芽孢杆菌中的多个调控系统协调 DNA 复制与细胞生长。

• DOI: http://dx.10.1371/journal.pgen.1004731
• 发表时间: 2014
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Murray H

Identification of a basal system for unwinding a bacterial chromosome origin.

鉴定用于解开细菌染色体起源的基础系统。

• DOI: http://dx.10.15252/embj.2019101649
• 发表时间: 2019
• 期刊: The EMBO journal
• 作者: Richardson TT

The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere.

压缩细菌着丝粒的动态 DNA 结合和桥接相互作用的结构基础。

• DOI: http://dx.10.7554/elife.28086
• 发表时间: 2017
• 期刊: eLife
• 影响因子: 7.7
• 作者: Fisher GL

Condensin- and Replication-Mediated Bacterial Chromosome Folding and Origin Condensation Revealed by Hi-C and Super-resolution Imaging.

Hi-C 和超分辨率成像揭示了凝缩和复制介导的细菌染色体折叠和起始凝缩。

• DOI: 10.1016/j.molcel.2015.07.020
• 发表时间: 2015-08-20
• 期刊: Molecular cell
• 影响因子: 16
• 作者: M. Marbouty;Antoine Le Gall;D. Cattoni;A. Cournac;Alan Koh;J. Fiche;J. Mozziconacci;H. Murray;R. Koszul;M. Nollmann
• 通讯作者: M. Nollmann