Investigating the roles of the ubiquitin ligases Cullin2\(^{Lrr1}\) and TRAIP during DNA replication termination

Poovathumkadavil, Divyasree ORCID: 0009-0004-4331-4728 (2024). Investigating the roles of the ubiquitin ligases Cullin2\(^{Lrr1}\) and TRAIP during DNA replication termination. University of Birmingham. Ph.D.

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Abstract

DNA replication is a well-orchestrated process occurring throughout the S-phase of the cell cycle and unperturbed replication is the basis for genome maintenance. To understand how genome integrity is maintained, it is critical to study the underlying mechanisms and regulation of DNA replication. The defects in the first two stages of DNA replication- initiation and elongation are known to contribute to human diseases and are already targeted by cancer therapies. Recent investigations suggest that disruption of the final stage of replication-termination can also lead to genomic instability. As replication reaches completion and replication forks converge, replisomes are unloaded from chromatin. This unloading mechanism is highly regulated to prevent the unregulated unloading of active replisomes.
Using Xenopus laevis egg extracts, the Gambus lab had previously provided the first insights into replisome disassembly mediated by Cullin2\(^{Lrr1}\) . This PhD project aimed to further understand the mechanism by which Cullin2\(^{Lrr1}\) identifies its substrate through structural analysis. While the purification of Lrr1 proved technically challenging, we employed in silico methods to gain high confidence structural predictions for Lrr1.
Furthermore, we have also identified the existence of a backup pathway for replisome disassembly in mitosis. This mitotic pathway uses a different E3 ubiquitin ligase - TRAIP. Our findings demonstrate that catalytically active TRAIP plays an essential role in post-termination and stalled replisome disassembly in mitosis. Following this, we also explored TRAIP’s involvement in replisome disassembly during S-phase. Intriguingly, the depletion of TRAIP did not affect replisome disassembly in S-phase within Xenopus laevis egg extracts. Moreover, this PhD project also indicates that the TRAIP activity in both S-phase and mitosis is subject to regulation via post-translational modifications,
specifically phosphorylation.
Therefore, this thesis expands our understanding of the mechanisms upholding genome stability during termination of DNA replication. It uncovered dual regulation of replisome removal in mitosis versus S-phase through activation of distinct ubiquitin-dependent pathways.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):