A distinct mode of DNA replication initiation in trypanosomes?

所有生物的生长和繁殖都需要基因组的忠实传递，因为这种遗传物质提供了生命的蓝图。基因组从父母传递给后代需要遗传物质被复制，然后被分离。在所有的细胞生物中，基因组都是由DNA组成的，它的复制被称为DNA复制。在真核细胞（如人类和酵母）中，DNA基因组的复制是一个高度调控的多步骤过程。DNA复制始于一种特定的蛋白质复合物，即“启动器”与基因组的特定区域的结合。这些区域被称为起源，是DNA复制开始的地方。启动物与起点的结合导致一系列事件，导致其他参与者被招募到起点，包括另一种称为“解旋酶”的蛋白质复合物；这个复合体在起始处打开DNA，并允许复制机器（例如DNA聚合酶）沿着DNA移动，复制每条链以产生两个几乎相同的原始DNA副本。然而，解旋酶不是由起始物直接招募到原点的，而是通过两种中介蛋白招募到原点的。在大多数已研究的真核生物中，引发剂、解旋酶和这两种介质都存在，并且非常相似。此外，所有这些蛋白质在细胞生长的特定阶段被招募到起源，在DNA复制发生之前。然而，在非洲锥虫中，DNA复制的这两个方面似乎是不同的。非洲锥虫是单细胞真核微生物，在撒哈拉以南非洲的人类和其他动物（如牛）中引起严重和衰弱性疾病。我们（和其他人）的工作结果表明了三件事：锥虫的引发物看起来与其他真核生物（人类、苍蝇和酵母）中看到的蛋白质复合物非常不同；目前尚不清楚这两种介质是否存在于这些微生物中；启动物，中介蛋白（如果存在的话）和解旋酶之间相互作用的时间发生在与其他真核生物不同的细胞生长阶段。在这个项目中，我们希望通过一系列的调查来探索上面提到的差异。首先，我们发现了一种与引发剂相互作用的蛋白质，但它与其他真核生物中的任何引发剂成分都不相似；我们想知道它是否是启动子的一部分，以及它是如何影响DNA复制的。其次，由于尚不清楚锥虫是否有这两种介质，我们想测试在锥虫中发现的两种蛋白质是否提供这些功能以及它们是如何做到的。最后，我们想要探索启动物、假定的中介蛋白和解旋酶之间相互作用时间的动力学，如上所述，解旋酶似乎发生在与其他真核生物不同的细胞生长阶段。为了进行这些分析，我们将使用广泛的细胞和遗传方法。如果我们的假设是正确的，我们的发现将改变对真核生物中DNA复制调控的主流观点，并开辟新的研究方向。此外，我们的工作将揭示DNA复制如何与锥虫体内广泛的其他细胞过程联系在一起，包括它们如何在宿主中生存和表达其基因组内容。重要的是，从长远来看，如果锥虫体内DNA复制的参与者确实与其宿主（人类和动物）中的参与者非常不同，我们的发现将为开发新的寄生虫病治疗方法提供一组潜在的靶点。

The growth and propagation of all living organisms requires the faithful transmission of their genome, as this genetic material provides the blueprint for life. Transmission of the genome from parent to offspring requires that the genetic material be copied and then segregated. In all cellular organisms the genome is composed of DNA, and its copying is referred to as DNA replication. In eukaryotic cells (e.g. humans and yeast), replication of the DNA genome is a highly regulated, multi-step process. DNA replication starts with the binding of a specific protein complex, the 'initiator', to specific regions of the genome. These regions are called origins and are the sites where copying of the DNA begins. Binding of the initiator to the origins leads to a series of events that result in the recruitment of other players to the origins, including another protein complex called the 'helicase'; this complex opens the DNA at the origin and allows the copying machinery (e.g. DNA polymerases) to travel along the DNA, copying each strand to generate two near-identical copies of the original DNA. The helicase is not, however, recruited to the origin directly by the initiator, but through two mediator proteins. In most eukaryotes organisms that have been studied, the initiator, helicase and the two mediators are present and are quite similar. Furthermore, all these proteins are recruited to the origins at a specific stage of the cell's growth, prior to the stage where DNA replication takes place. In African trypanosomes, however, both these aspects of DNA replication seem to be different. African trypanosomes are single cell eukaryote microbes that cause severe and debilitating disease in both humans and other animals (such as cattle) in sub-Saharan Africa. The results of our work (and from others) suggest three things: that the trypanosome's initiator looks very different from the protein complex seen in other eukaryote organisms (human, fly and yeast); it is unclear if the two mediators exist in these microbes; and that the timing of interaction between the initiator, the mediator proteins (if they exist) and the helicase occurs at a distinct stage of cell growth from that seen in other eukaryotes. With this project, we want to explore the differences mentioned above by following a number of lines of investigation. First, we have found a protein that interacts with the initiator, but it is not similar to any of the initiator components in other eukaryotes; we want to find out if it is part of the initiator and how it affects DNA replication. Second, as it is unclear if trypanosomes have the two mediators, we want to test if two proteins identified in trypanosomes provide these functions and how do they do so. Finally, we want to explore the dynamics of the timing of interaction between the initiator, the putative mediator proteins, and the helicase which, as mentioned above, seems to take place at a different stage of cell growth from other eukaryotes. To perform these analyses, we will use a wide range of cellular and genetic approaches. If our hypotheses are correct, our findings will alter the prevailing view of DNA replication regulation across the diverse range of eukaryotic organisms, and open new lines of research. In addition, our work will reveal how DNA replication connects with a wide range of other cellular processes in trypanosomes, including how they survive in the host and express the content of their genome. Importantly, and in the long term, if the players in DNA replication in trypanosomes are indeed very different from those in their hosts (humans and animals), our findings will provide a group of potential targets for the development new treatments for parasitic diseases.