A distinct mode of DNA replication initiation in trypanosomes?

锥虫中 DNA 复制起始的独特模式？

• 批准号: BB/W001101/1
• 负责人: Richard McCulloch
• 金额: $ 96.44万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW001101%2F1
• 关键词: distinct; mode; DNA; replication; initiation

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.

所有生物体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。基因组从父母传递到后代需要复制遗传物质，然后分离。在所有细胞生物中，基因组由DNA组成，其复制被称为DNA复制。在真核细胞（例如人类和酵母）中，DNA基因组的复制是一个高度调控的多步骤过程。DNA复制开始于一个特定的蛋白质复合物的结合，“启动子”，基因组的特定区域。这些区域被称为起源，是DNA复制开始的地方。引发剂与起始点的结合导致一系列事件，导致其他参与者被招募到起始点，包括另一种称为“解旋酶”的蛋白质复合物;这种复合物在起始点打开DNA，并允许复制机器（例如DNA聚合酶）沿着DNA沿着，复制每条链以产生原始DNA的两个几乎相同的拷贝。然而，解旋酶不是直接由起始物募集到起始处，而是通过两个介体蛋白。在已经研究的大多数真核生物中，存在起始剂、解旋酶和两种介质，并且它们非常相似。此外，所有这些蛋白质在细胞生长的特定阶段被募集到起点，在DNA复制发生的阶段之前。然而，在非洲锥虫中，DNA复制的这两个方面似乎不同。非洲锥虫是单细胞真核生物微生物，在撒哈拉以南非洲的人类和其他动物（如牛）中引起严重和衰弱的疾病。我们的工作成果（和其他人）提出了三件事：锥虫的起始物看起来与其他真核生物中看到的蛋白质复合体非常不同（人类、苍蝇和酵母）;目前还不清楚这两种介质是否存在于这些微生物中;并且发起者之间的交互的定时，介体蛋白（如果它们存在的话）和解旋酶发生在与其它真核生物中所见的不同的细胞生长阶段。在这个项目中，我们希望通过以下几条调查路线来探索上述差异。首先，我们发现了一种与启动子相互作用的蛋白质，但它与其他真核生物中的任何启动子组分都不相似;我们想知道它是否是启动子的一部分，以及它如何影响DNA复制。其次，由于目前还不清楚锥虫是否具有这两种介质，我们想测试锥虫中鉴定的两种蛋白质是否提供这些功能以及它们是如何做到这一点的。最后，我们要探索的动力学之间的相互作用的启动子，推定的介质蛋白，和解旋酶，如上所述，似乎发生在不同的阶段，从其他真核生物的细胞生长的时间。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DNA复制调控在各种真核生物中的流行观点，并开辟新的研究方向。此外，我们的工作将揭示DNA复制如何与锥虫中的其他细胞过程联系在一起，包括它们如何在宿主中生存并表达其基因组的内容。重要的是，从长远来看，如果锥虫中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寄生虫病的新疗法提供一组潜在靶点。

项目成果

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

• DOI: 10.1093/nar/gkad836
• 发表时间: 2023-11-10
• 期刊: Nucleic acids research
• 影响因子: 14.9