Investigating the role of DTX3L in the replication stress response
Thesis or Dissertation
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DNA replication must be carried out with high fidelity to ensure accurate transfer of genetic information upon cell division. The mechanisms by which damage upon DNA is repaired are extensive and are increasingly well characterised; in contrast, the mechanisms that maintain DNA replication in the face of these challenges, known as the replication stress response (RSR), are less well understood. Cancer cells often harbour mutations in these pathways to enable carcinogenesis; conversely, a reliance on remaining pathways provides opportunity for their selective targeting to elicit genomic instability and cancer cell death. Therefore, the identification of novel factors of the RSR will provide new opportunities for drug development. Through the use of isolation of proteins on nascent DNA (iPOND), E3 ubiquitin ligase DTX3L has been identified as a novel factor present at the progressing replisome. DNA fibre analysis has suggested DTX3L to be an important factor in maintaining replication fork dynamics and resection. Quantification of DNA damage response (DDR) markers suggests that altered replication dynamics as a result of DTX3L depletion are consequential, resulting in increased genomic instability. Furthermore, an enhanced sensitivity to clastogen treatment in DTX3L deficient cells is observed, demonstrating clinical relevance. Mechanistically, through abrogation of ubiquitin ligase domain functionality, DTX3L ubiquitination is essential for its replication stress associated role. Evidence presented here suggests that DTX3L depletion may result in defective ATR activation and this may be mediated through DTX3L association with TOPBP1. Furthermore, ubiquitome enrichment analysis reveals other potential targets of DTX3L ubiquitination that may contribute to its role in maintenance of DNA replication. Ultimately, the work here evidences DTX3L as a newly identified factor of the RSR.
Cancer and Genome Instab
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Institute of Cancer Research (University Of London)