TRAIP ubiquitin ligase driving replisome disassembly

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.

Cell division requires the accurate duplication of all genetic information. DNA replication is precisely regulated as errors can lead to severe consequences, such as genetic disease, neurodegeneration and premature ageing. Surprisingly, despite six decades of research, little was known about the completion of eukaryotic replication, until a few years ago when we described the first elements of the eukaryotic replisome disassembly mechanism. Now we have discovered the existence of a second pathway of replisome unloading: acting in mitosis and driven by TRAIP ubiquitin ligase. This novel pathway leads to unloading of not only the post-termination replisomes but also stalled replisomes retained on replication forks until mitosis. Unloading of such replisomes is likely to be essential for mitotic processing of under-replicated DNA associated with them through mitotic DNA synthesis and ultrafine bridges in order to maintain genomic stability. TRAIP ubiquitin ligase is also known to be important for resolution of DNA replication stress in human cells, but its substrates are unknown. This proposal aims to establish that the replicative helicase is a direct substrate of TRAIP ubiquitylation that leads to replisome unloading and to characterise the activity of TRAIP ubiquitin ligase. We will also elucidate how TRAIP is regulated and activated to unload replisomes only at the right time. Uncontrolled replisome dissolution would be catastrophic for cell viability and genomic stability. Based on our preliminary data we hypothesise that TRAIP is activated through phosphorylation specifically in mitosis.
We will deliver our objectives in a two-pronged approach: (i) through detailed biochemical analysis of the process in a cell-free Xenopus laevis egg extract system, followed by (ii)
cell biology validation of the process and investigation into the consequences of its disruption in human immortalised cell lines and patient samples.

Outcomes arising from this investigation will have a direct impact at both national and international level. Our project is positioned at the fundamental stage of knowledge generation and will enhance our understanding of the essential process of DNA replication and cellular pathways preventing genomic instabilities, but also provide insight into the development and treatment of neurodegenerative disorders, cancer and ageing processes.
Generated outcomes will be far-reaching and beneficial to a broad cross-section of academics and scientists with interests spanning from DNA replication and replication stress, post-translational modification of proteins, genome stability and the Xenopus egg extract as a model system. Biomedical scientists and clinicians with interests in chromosomal instability (CIN) generation (cancer development, ageing and neurodegeneration) should also find commonalities and crossovers with their studies (outlined in details in Academic Beneficiaries section).
This project will also have a direct impact on the careers of my trained postdoctoral researcher, technician and postgraduate and undergraduate students. It will lead to employment and training of two researchers and allow us to continue training PhD students, Masters and undergraduate students. Training of new generations of scientists is very important to my laboratory and we enjoy enthusing young people.

Public beneficiaries of this research will include patients suffering from genetic disorders caused by mutations in TRAIP and other replication stress factors, cancer patients, rare disease charities and biotechnology companies.
1. Patients suffering from genetic disorders caused by mutations in TRAIP (Seckel syndrome) and other replication and replication stress factors.
We will investigate the function and regulation of ubiquitin ligase TRAIP that when mutated causes Seckel Syndrome, which manifests as primordial dwarfism. Our investigations have great potential for shedding new light onto the mechanism of generation of the phenotype observed in Seckel syndrome patients but also of a growing number of genetic disorders resulting from mutations in DNA replication factors. Better knowledge of the function of TRAIP will increase our understanding of the development of these disorders and may suggest novel ways to help patients with conditions caused by such mutations.
2. Cancer patients.
Improved treatments for cancer are of general interest among the public but especially among the cancer sufferers. To achieve this goal we need to better understand the ways in which cancer develops, the mechanism by which current therapies work to improve them and widen our knowledge of processes essential for cancer phenotypes, which can be targeted by new drugs. Many cancer patients are treated with radiotherapy and chemotherapy targeting DNA replication which, although effective, are dose-limiting and cause many side effects. It is therefore of great interest to identify new targets affecting essential processes for cancer development and growth such as DNA replication, which will be as effective as current ones but much more cancer-specific. We plan to determine the mechanism of a still relatively unknown stage of DNA replication and genome maintenance processes, which may provide future therapy targets.
3. The NHS, rare disease charities and pharmaceutical companies.
All of these organisations seek development of better and more specific drugs. Our proposed research focuses on determining the molecular regulation of TRAIP ubiquitin ligase and so has the potential to benefit them all. Moreover, we will share our enthusiasm and dedication for science with representatives of cancer charities and fundraisers to show appreciation for their hard work. We will also collaborate with AstraZeneca to facilitate the translational aspect of our research.