Assembly of the bacterial DNA replication initiation complex

细胞是所有生物体的基本结构单位。细胞要生长和分裂，必须遵循一个蓝图，该蓝图提供了描述如何进行这些基本活动的说明。在所有细胞中，这些信息都被编码在DNA中。每次细胞分裂时，它必须复制自己的DNA，并将一个完整的、完好无损的拷贝传递给每一个后代细胞。DNA复制必须严格控制，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完整的遗传信息，将无法存活。如果DNA复制发生得比需要的早，细胞就会包含太多的基因拷贝，导致表达水平的改变，从而导致发育缺陷。DNA复制的开始需要专门的复制启动蛋白。启动蛋白结合到称为复制起点的位点，在那里它们的作用是招募DNA复制机制。在生命的三个领域中，所有的启动蛋白都含有一个相关的蛋白折叠（启动特异性的AAA+基序），这表明它们具有共同的活性所需的活性。细菌的结构和生理特征相对简单，是研究DNA复制分子机制的理想系统，因为它们易于进行遗传操作，而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靶标，因为它对生长至关重要，而且目前不存在预先存在的耐药性问题。细菌DNA复制启动蛋白被称为DNA。DNA与细菌复制起点内的特定序列结合，形成一个大的核蛋白复合体，分离DNA双链的两条链，启动基因组复制过程。令人兴奋的是，dna打开复制起点的分子基础开始出现。使用x射线晶体学的结构研究发现，DNA组装成螺旋状细丝，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获得DNA活性的分子理解是必要的，但重要的是要注意它们是有限的，因为它们只提供了动态DNA复制起始反应的静态图像。此外，DNA在复制起始DNA的存在下不会结晶。因此，DNA的结构并不能揭示蛋白质最初是如何在复制起点组装成寡聚物的，也不能揭示DNA是如何转变成被认为可以拉伸和打开DNA的构象的。我的实验室以前的工作已经建立了一种新的生化分析方法，使用纯化的蛋白质来检测dna螺旋的形成。我们最近改进了这种方法，现在能够证明dna在复制起源处至少采用两种不同的螺旋组装状态。本研究的目的是通过鉴定DNA复制起源中组装初始DNA螺旋所需的序列和促进不同DNA构象之间转换所需的序列，来研究DNA复制起始的途径。我们将利用遗传学方法来解剖体外和体内的起源区域，然后我们将利用我们新的螺旋形成分析来确定这些变化如何影响dna。该项目将提供尖端的知识，并将支持未来关于细胞活力和增殖至关重要的基本生物学问题的研究。

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.