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The Identification and Characterisation of Novel Suppressors of DNA Replication Stress

Tripp, Charlotte E (2017) The Identification and Characterisation of Novel Suppressors of DNA Replication Stress. PhD thesis, University of Sheffield.

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Abstract

Replication stress is present in cancers cells at higher than normal levels due to the increased proliferation caused by oncogene activation. This results in elevated levels of phosphorylated RPA2 (pRPA2) and DNA damage, which can ultimately result in genetic instability. By inhibiting suppressors of replication stress, it may be possible to target this cancer-specific phenotype to selectively kill tumour cells. Attempts were therefore made to develop a high throughput whole genome RNAi screen to detect increased endogenous levels of pRPA2 foci formation following gene knockdown, however, this proved unsuccessful. As a contingency measure a selected panel of kinases, identified as putative replication stress suppressors and potential druggable targets, were assessed for their ability to induce pRPA2 foci following gene knockdown. This approach identified several hits, which were then evaluated for their ability to preferentially kill cells lacking p53 signalling or overexpressing several clinically relevant oncogenes (Cyclin E, H-RAS and MYC-N). They were also assessed for their ability to potentiate the effects of the replication stress inducing drugs Gemcitabine and 5-Flurouracil as well as the PARP inhibitor Olaparib. In addition, the putative DNA damage repair factor CCDC15, which has been hypothesised to act in the resolution of replication impeding lesions, was also investigated as a potential replication stress suppressor. However, it does not appear to modulate DNA replication stress as its loss does not induce pRPA2 and the formation of DNA damage following its knockdown is independent of entry into S phase. Interestingly, depletion of CCDC15 sensitised certain cell lines to the effects of DNA crosslinking agents. Furthermore, knockdown of CCDC15 also slightly increased the formation of endogenous FANCD2 foci, but not in cells treated with DNA crosslinking agents. However, CCDC15-depleted cells exhibited delayed formation and resolution of RAD51 foci following DNA damage, which were not due to perturbations in cell cycle progression. Although CCDC15 appears to have little effect on the cell cycle distribution of cycling cells, its loss potentially delays re-entry into the cycle of cells paused in G1 phase.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.741208
Depositing User: Miss Charlotte E Tripp
Date Deposited: 21 May 2018 08:28
Last Modified: 01 Jun 2020 09:53
URI: http://etheses.whiterose.ac.uk/id/eprint/20385

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