TRAIP ubiquitin ligase driving replisome disassembly

自1953年沃森和克里克提出他们的DNA结构模型并提出半保存复制的优雅方式以来，DNA复制的过程一直被研究。令人惊讶的是，尽管经过了60年的研究，人们对真核生物复制的完成却知之甚少。2014年，我的团队发现了DNA复制过程中复制机械解体机制的第一个要素，为这一领域提供了急需的突破。然而，我们的新数据表明，复制机制的移除可以通过两种途径完成：一种发生在DNA复制期间，另一种途径发生在细胞周期的下一阶段，即有丝分裂，当细胞试图分裂其DNA时。有趣的是，在有丝分裂中起作用的途径可以从DNA中卸载任何复制机制，包括那些由于未完成的复制或复制问题而停滞的复制机制。这种未完成的复制在癌细胞有丝分裂中经常观察到。我们认为，移除不适当地粘在DNA上的复制机制对于DNA周围的加工和保持遗传物质的稳定性至关重要。众所周知，DNA复制过程中的问题可能导致突变和染色体改变，从而导致癌症的发展、过早衰老和神经退行性变。因此，理解正确删除复制机制如何保护我们免受此类问题的困扰是很重要的。我们发现有丝分裂中复制机制的卸载取决于一种特定酶的活性：TRAIP泛素连接酶。众所周知，TRAIP对于解决DNA复制过程中出现的问题非常重要，而人类细胞中TRAIP的突变导致了原始侏儒症。这一提议旨在确定复制解旋酶（解绕DNA的蛋白质复合体和复制机制的组织中心）确实是TRAIP修饰的底物。我们还将阐明TRAIP连接酶活性的模式。最后，我们希望了解如何激活TRAIP以仅卸载不再需要的复制机器。对细胞来说，不受控制的复制机制的解体将是灾难性的，因为它将阻止它们完成基因组复制。我们认为TRAIP的激活是通过TRAIP自身的有丝分裂特异性修饰实现的。为了实现我们的目标，我们将使用无细胞非洲爪蟾卵提取物模型系统和永生人类细胞系的细胞生物学结合生化方法。卵子提取系统提供了一个简化的模型，其中DNA复制发生在试管中，与大多数其他细胞过程分离。重要的是，这个复制过程就像在细胞中一样受到调节，许多关键的和开创性的复制发现都是利用这个系统进行的。鸡蛋提取物使我们能够对我们正在研究的新机制提出精确而具体的问题。我们将使用人类永生化细胞系和从携带TRAIP突变的患者样本中建立的原代细胞系来证实我们在非洲爪蟾系统中的发现。这个项目的结果将回答关于DNA复制过程的基本问题，重点是高度未被研究的终止阶段。我们将提供一种理解，即必不可少的泛素连接酶TRAIP的调控在复制机制的拆卸和表征新的复制因子中起着关键作用。了解TRAIP的调控和激活将大大增加我们对维持基因组稳定性的DNA复制问题的细胞保护途径的认识。

The process of DNA replication has been studied ever since Watson and Crick presented their model of the DNA structure in 1953 and proposed the elegant way of semiconservative replication. Surprisingly, despite six decades of research, little was known about the completion of eukaryotic replication. In 2014 my group discovered the first elements of the mechanism of replication machinery disassembly during DNA replication, providing the much-needed breakthrough in this field. However, our new data show that the removal of the replication machinery can be accomplished by two pathways: one occurring during DNA replication and a second pathway in the next stage of the cell cycle, mitosis, when cells try to divide their DNA. Interestingly, the pathway acting in mitosis can unload any replication machinery from DNA, including those stalled due to unfinished replication or problems with replication. Such unfinished replication is often observed in mitosis in cancer cells. We believe that removal of replication machineries inappropriately stuck on DNA is essential for processing of DNA around it and retains stability of the genetic material. It is well established that problems during DNA replication can lead to mutations and chromosomal changes driving cancer development, premature aging and neurodegeneration. It is important therefore to understand how correct removal of replication machineries can protect us from such problems. We have discovered that the unloading of replication machineries in mitosis depends on activity of a specific enzyme: TRAIP ubiquitin ligase. TRAIP is known to be important for resolution of problems arising during DNA replication and mutations in TRAIP in human cells leads to primordial dwarfism. This proposal aims to establish that the replicative helicase (the protein complex unwinding DNA and the organising center of the replication machinery) is indeed the substrate modified by TRAIP. We will also elucidate the mode of TRAIP's ligase activity. Finally, we wish to understand how TRAIP is activated to unload only replication machineries that are not needed anymore. Uncontrolled dissolution of acting replication machineries would be disastrous for cells as it would stop them from completing genome replication. We believe that activation of TRAIP is achieved through mitosis-specific modification of TRAIP itself.To achieve our aims, we will combine biochemical approaches using the cell-free Xenopus laevis egg extract model system and cell biology in immortalised human cell lines. The egg extract system provides a simplified model, where DNA replication happens in a tube in separation from most of the other cellular processes. Importantly, this process of replication is regulated just like in cells and many key and seminal replication discoveries were made using this system. Egg extract allows us to ask precise and specific questions about the novel mechanisms we are investigating. We will confirm our findings made in the Xenopus system using human immortalised cell lines and primary cell lines established from patient samples with TRAIP mutations.The results of this project will answer fundamental questions about the process of DNA replication, focusing on the highly understudied termination stage. We will deliver an understanding that regulation of the essential ubiquitin ligase TRAIP plays a key role in replication machinery disassembly and characterise novel replication factors. Understanding the regulation and activation of TRAIP will significantly increase our knowledge of the cellular pathways of protection from DNA replication problems in the maintenance of genome stability.