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。细胞生长和分裂必须遵循一个蓝图，该蓝图提供了描述如何执行这些基本活动的指令。在所有细胞中，这些信息都编码在DNA中。每次细胞分裂时，它必须复制它的DNA，并将一个完整的、未受损的拷贝传递给每个后代细胞。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，一个子细胞将不能继承完整的遗传信息，并且将是不可存活的。如果DNA复制发生早于所需，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复制的开始需要专门的复制起始蛋白。起始蛋白结合到称为复制起点的位点，在那里它们起作用以募集DNA复制机器。在整个生命的三个王国中，所有起始蛋白都包含相关的蛋白质折叠（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良好的结构和生理学，是研究DNA复制的分子机制的理想系统，因为它们易于进行遗传操作，并且它们的蛋白质往往是生物化学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点，因为它对生长至关重要，并且目前没有预先存在的耐药性问题。DnaA与细菌复制起点内的特定序列结合，并形成一个大的核蛋白复合物，该复合物将DNA双链体的两条链分开，以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获得对DnaA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，因为它们仅提供动态DNA复制起始反应的静态图像。此外，DnaA在复制起点DNA的存在下不结晶。因此，DnaA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体的，也不能揭示DnaA是如何转变成被认为是拉伸和打开DNA的构象的。在我的实验室以前的工作已经建立了一个新的生化分析使用纯化的蛋白质，检测DnaA螺旋的形成。我们最近改进了这一方法，现在能够证明DnaA在复制起点处采用至少两种不同的螺旋装配状态。本研究项目的目的是通过鉴定复制起点内组装初始DnaA螺旋所需的序列和促进DNA复制起始点之间的转换所需的序列来研究DNA复制起始的途径。不同的DNA构象我们将利用遗传学方法在体外和体内解剖起源区域，然后我们将利用我们的新的螺旋形成试验来确定这些变化如何影响DnaA。该项目将提供尖端知识，并将支持未来关于细胞活力和增殖所必需的基本生物学问题的研究。

项目成果

The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

细菌 DnaA-trio 复制起点元件指定单链 DNA 起始子结合。

• DOI: 10.1038/nature17962
• 发表时间: 2016-06-16
• 期刊: Nature
• 影响因子: 64.8
• 作者: Richardson TT;Harran O;Murray H
• 通讯作者: Murray H

Erratum: The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

勘误表：细菌 DnaA-trio 复制起点元件指定单链 DNA 起始子结合。

• DOI: 10.1038/nature18932
• 发表时间: 2016
• 期刊: Nature
• 影响因子: 64.8
• 作者: Richardson TT

Probing Chromosome Dynamics in Bacillus subtilis.

探索枯草芽孢杆菌的染色体动力学。

• DOI: 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.

多个调节系统将DNA复制与枯草芽孢杆菌中的细胞生长进行协调。

• DOI: 10.1371/journal.pgen.1004731
• 发表时间: 2014-10
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Murray H;Koh A
• 通讯作者: Koh A

Crystal structure of NucB, a biofilm-degrading endonuclease.

• DOI: 10.1093/nar/gkx1170
• 发表时间: 2018-01-09
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Baslé A;Hewitt L;Koh A;Lamb HK;Thompson P;Burgess JG;Hall MJ;Hawkins AR;Murray H;Lewis RJ
• 通讯作者: Lewis RJ