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质（“启动者”）在特定位点（“起始点”）解开DNA双螺旋。DNA双解绕提供了单链的复制机制，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都是高度保守的，并且具有很好的特征，但起源是谜。事实上，在像人类这样的高等生物中，我们还没有定义是什么构成了起源。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。最近，我们报道了一种新的复制起始元件（称为“DNA -trio”）的鉴定，并表明它可以促进主细菌复制启动蛋白DnaA的单链DNA结合活性。我们提出，这一重要的识别元件代表了核心细菌染色体起源的保守成分。这一提议的目标之一是确定dna三重奏在细菌王国中有多普遍。我们特别感兴趣的是测试这种染色体起源元素是否存在并活跃于人类病原体中，如金黄色葡萄球菌和幽门螺杆菌，因为这将确定抗生素开发的新靶点。另一个目标是表征dna -trios与启动蛋白dna的相互作用。进一步了解dna识别的分子机制和这种新的染色体起源元件的活性，将为生存和增殖所必需的基本生物学过程提供前沿知识。值得强调的是，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质折叠（启动特异性的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动。因此，这个研究项目的发现将告诉我们真核启动蛋白是如何起作用的，并将支持在高等生物中寻找复制起源元素，这在目前是不明确的。

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.