TRAIP ubiquitin ligase driving replisome disassembly

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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.