Understanding the essential role of the E3 ubiquitin ligase TRAIP during the eukaryotic cell cycle

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Scaramuzza, Shaun ORCID: https://orcid.org/0000-0001-8470-6462 (2022). Understanding the essential role of the E3 ubiquitin ligase TRAIP during the eukaryotic cell cycle. University of Birmingham. Ph.D.

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Abstract

The RING-type E3 ubiquitin ligase TRAIP has been widely associated with the promotion of genomic stability throughout the cell cycle. In addition, TRAIP has been linked with several aspects of health and disease; mutations to TRAIP have been reported to lead to primordial dwarfism in patients and TRAIP is differentially expressed across several forms of cancer. However, despite this, the essential role of TRAIP during the otherwise unperturbed cell cycle, responsible for the associated cell lethality and primordial dwarfism phenotypes, remained unclear.

This PhD project aimed to understand further the roles of TRAIP during individual stages of the cell cycle. Through the generation of conditional degron cell lines, we demonstrate that the essential role for TRAIP occurs specifically during S-phase. Further work then identified the likely underlying mechanism of action: the resolution of damaging DNA replication - transcription conflicts. As a result, without TRAIP, cells accumulate DNA damage throughout S-phase, arrest in G2 due to ATR checkpoint activation, and ultimately exit the cell cycle via senescence.

Following the characterisation of these S-phase mechanisms involving TRAIP, we next explored any requirements during mitosis. The Gambus lab had previously found TRAIP to accumulate with the terminating replisome, upon inhibition of the S-phase replisome disassembly pathway. Subsequent work then revealed the ability of TRAIP to drive mitotic replisome disassembly in X. laevis egg extract. In this project, we demonstrate that the core mechanisms of replisome disassembly, described using X. laevis egg extract, C. elegans, and S. cerevisiae, are likely conserved into human somatic cells. Moreover, we present evidence to suggest that TRAIP can also drive mitotic replisome disassembly in mammalian cell lines. Finally, TRAIP was shown to be important for mitotic timing, independently of any S-phase roles.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):