Investigating the DnaA-trio, a new essential bacterial replication origin element that specifies single-stranded DNA initiator binding

在所有细胞中，基因组复制都需要关键蛋白（“启动子”）在特定位点（“起点”）解开DNA双螺旋。DNA双链体解旋为复制机制提供了进入单链的途径，单链作为新一轮DNA合成的模板。虽然引发剂在所有生物体中高度保守，并且具有很好的特征，但起源是神秘的。事实上，在高等生物体中，例如人类，我们还没有定义什么构成了起源。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。最近，我们报道了一种新的复制起始元件（称为“DnaA-三重奏”）的鉴定，并表明它的作用，以促进主细菌复制起始蛋白，DnaA的单链DNA结合活性。我们提出，这个基本的识别元件代表了核心细菌染色体起源的保守组成部分。这项计划的目标之一是确定DnaA-trio在细菌王国中的分布范围。我们特别感兴趣的是测试这种染色体起源元件是否存在于人类病原体（如金黄色葡萄球菌和幽门螺杆菌）中并具有活性，因为这将确定抗生素开发的新靶点。另一个目标是表征DnaA-trios与起始蛋白DnaA的相互作用。进一步了解DnaA识别和活性与这个新的染色体起源元素的分子机制，将提供关于一个基本的生物过程，这是至关重要的生存能力和增殖的前沿知识。重要的是要强调的是，在生命的三个王国中，所有的起始蛋白都包含一个相关的蛋白质折叠（起始蛋白特异性AAA+基序），这表明它们是从一个共同的祖先进化而来的，并且它们具有共同的活性。因此，这项研究项目的发现将告知真核生物起始蛋白如何起作用，并将支持在高等生物中寻找复制起始元件，目前尚不清楚。

In all cells genome duplication requires key proteins ("initiators") to unwind the DNA double helix at specific sites ("origins"). DNA duplex unwinding provides the replication machinery access to single strands, which act as templates for new rounds of DNA synthesis. While initiators are highly conserved throughout all organisms and well characterised, origins are enigmatic. In fact within higher organisms, such as humans, we have yet to define what constitutes an origin. 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. Recently we reported the identification of a novel replication origin element (termed the "DnaA-trio") and showed that it acts to promote single-strand DNA binding activity of the master bacterial replication initiator protein, DnaA. We proposed that this essential recognition element represents a conserved component of the core bacterial chromosome origin. One of the goals of this proposal is to determine how widespread the DnaA-trio is within the bacterial kingdom. We are particularly interested in testing whether this chromosome origin element is present and active in human pathogens such as Staphylococcus aureus and Helicobacter pylori, since this would identify a novel target for antibiotic development. Another goal is to characterize the interaction of DnaA-trios with the initiator protein DnaA. Further understanding of the molecular mechanisms underlying DnaA recognition and activity with this new chromosome origin element will provide cutting-edge knowledge regarding a fundamental biological process that is essential for viability and proliferation. It is important to stress that all initiator proteins throughout the three kingdoms of life contain a related protein fold (the initiator specific AAA+ motif), indicating that they evolved from a common ancestor and that they share common activities. Therefore, the findings from this research project will inform how eukaryotic initiator proteins act and will underpin the search for replication origin elements in higher organisms, which at this moment are ill defined.