TRAIP ubiquitin ligase driving replisome disassembly

TRAIP 泛素连接酶驱动复制体拆卸

• 批准号: BB/T001860/1
• 负责人: Agnieszka Gambus
• 金额: $ 66.84万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT001860%2F1
• 关键词: TRAIP; ubiquitin; ligase; driving; replisome

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.

自1953年沃森和克里克提出DNA结构模型并提出保守复制的优雅方法以来，DNA复制过程一直受到人们的关注。令人惊讶的是，尽管研究了60年，但对真核生物复制的完成知之甚少。2014年，我的团队发现了DNA复制过程中复制机器分解机制的第一个元素，为该领域提供了急需的突破。然而，我们的新数据表明，复制机制的去除可以通过两种途径来完成：一种发生在DNA复制过程中，另一种发生在细胞周期的下一阶段，即有丝分裂，当细胞试图分裂DNA时。有趣的是，在有丝分裂中起作用的通路可以从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。这种未完成的复制经常在癌细胞的有丝分裂中观察到。我们认为，去除不适当地粘在DNA上的复制机器对于处理其周围的DNA并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变化，从而导致癌症发展、过早衰老和神经退行性变。因此，重要的是要了解如何正确删除复制机器可以保护我们免受此类问题。我们已经发现，在有丝分裂中复制机制的卸载依赖于一种特定酶的活性：TRAIP泛素连接酶。已知TRAIP对于解决DNA复制过程中出现的问题是重要的，并且人类细胞中TRAIP的突变导致原始侏儒症。该提议旨在确定复制解旋酶（蛋白质复合物解旋DNA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的连接酶活性的模式。最后，我们希望了解TRAIP是如何激活的，以便只卸载不再需要的复制机器。不受控制地溶解正在起作用的复制机器对细胞来说将是灾难性的，因为它将阻止它们完成基因组复制。我们相信TRAIP的激活是通过对TRAIP本身的有丝分裂特异性修饰来实现的。为了实现我们的目标，我们将联合收割机生物化学方法与永生化人类细胞系中的细胞生物学方法相结合，使用无细胞非洲爪蟾卵提取物模型系统。鸡蛋提取物系统提供了一个简化的模型，其中DNA复制发生在与大多数其他细胞过程分离的试管中。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使用这个系统进行的。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和具体的问题。我们将使用人类永生化细胞系和从TRAIP突变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。该项目的结果将回答有关DNA复制过程的基本问题，重点是高度欠研究的终止阶段。我们将提供一种理解，即调控的必要泛素连接酶TRAIP在复制机制拆卸和复制新的复制因子中起着关键作用。了解TRAIP的调节和激活将显着增加我们的知识，从DNA复制问题的细胞途径的保护，在维持基因组的稳定性。

项目成果

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation.

• DOI: 10.1093/nar/gkad694
• 发表时间: 2023-10-13
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Kingsley, Georgia;Skagia, Aggeliki;Passaretti, Paolo;Fernandez-Cuesta, Cyntia;Reynolds-Winczura, Alicja;Koscielniak, Kinga;Gambus, Agnieszka
• 通讯作者: Gambus, Agnieszka

The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase.

• DOI: 10.1016/j.jbc.2022.102234
• 发表时间: 2022-08
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Tarcan, Zeynep;Poovathumkadavil, Divyasree;Skagia, Aggeliki;Gambus, Agnieszka
• 通讯作者: Gambus, Agnieszka

The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

p97 辅因子 Ubxn7 促进 S 期复制体分解

• DOI: 10.1101/2021.12.16.472925
• 发表时间: 2021
• 作者: Tarcan Z

The structural mechanism of dimeric DONSON in replicative helicase activation

• DOI: 10.1016/j.molcel.2023.09.029
• 发表时间: 2023-11-16
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Cvetkovic, Milos A.;Passaretti, Paolo;Costa, Alessandro
• 通讯作者: Costa, Alessandro

TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells.

• DOI: 10.1038/s41467-023-40695-y
• 发表时间: 2023-08-21
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Scaramuzza, Shaun;Jones, Rebecca M. M.;Sadurni, Martina Muste;Reynolds-Winczura, Alicja;Poovathumkadavil, Divyasree;Farrell, Abigail;Natsume, Toyoaki;Rojas, Patricia;Cuesta, Cyntia Fernandez;Kanemaki, Masato T. T.;Saponaro, Marco;Gambus, Agnieszka
• 通讯作者: Gambus, Agnieszka