The role of nucleotide excision repair factors and the Mre11 nuclease in nucleoside analogue sensitivity

Beardmore, Richard (2015) The role of nucleotide excision repair factors and the Mre11 nuclease in nucleoside analogue sensitivity. PhD thesis, Prifysgol Bangor University.

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The Nucleoside Analogue (NA) gemcitabine (2', 2'-difluorodeoxycytidine; dFdC) is a clinically important cancer drug that is used in the treatment of solid tumours including pancreatic, metastatic breast, ovarian and non-small cell lung cancer (Bergman et al., 2002). Gemcitabine is similar to deoxycytidine (the naturally occurring nucleotide), in that after phosphorylation to its triphosphate form, dFdCTP competes with dCTP for integration into DNA during DNA replication and acts as a chain terminator, inhibiting further DNA synthesis (Prakasha Gowda et al., 2010). For a cell to survive treatment with this drug, the modified nucleotide needs to be removed from the DNA to allow replication restart. It remains largely unknown which DNA repair pathways are responsible for the removal of NAs from DNA. Based on previous data from the Hartsuiker lab using the model organism Schizosaccharomyces pombe (S. pombe), it was hypothesised that the Nucleotide Excision Repair (NER) pathway may be one mechanism that is used for gemcitabine removal from the genome. NER is responsible for removing bulky adducts from the DNA strand, particularly those caused by UV radiation, such as 6-4 photoproducts, or cyclopyrimidine dimers (Vermeulen, 2011). NER is an orchestrated process which is carried out by no less than 30 proteins, with a core set of 7 proteins, termed XPA to G, that are responsible for the main mechanism. The whole NER mechanism can be broken down into the steps of recognition of damage, DNA unwinding, confirmation of damage, excision of the lesion, followed by repair and ligation of the strand. NER can be further categorised into global NER (GG-NER) where damage is recognised by surveillance of the whole genome, or transcription coupled NER (TC-NER) where the damage is detected by the stalling of RNA polymerase, in transcription (Kamileri et al., 2012). Genetic deficiency in NER causes the condition termed Xeroderma Pigmentosum (XP). XP patients have extreme sensitivity to UV light and have a 1000 fold increased incidence of skin cancers if exposed to sunlight (Ahmad and Hanaoka, 2008; Rezvani et al., 2010). Work here uses fibroblasts from XP patients to investigate the mechanisms within NER, in both structural and enzymatic requirements of each NER protein, to determine their role in the removal of nucleoside analogues from the DNA. The resistance of some cancers to chemotherapeutic attack is often a result of the work done by such DNA repair pathways, therefore understanding the repair pathway that offers resistance to various drugs is essential for optimising treatment for individual cancers. Results presented in this thesis show that NER factors are required for cell survival following nucleoside analogue treatment, particularly XPA, XPC and XPG. Phenotypes shown following nucleoside analogue treatments in various NER deficient cell lines from different XP complementation groups differ from the classical reported sensitivity to UV and the UV mimetic drug 4NQO as measured by colony formation and unscheduled DNA synthesis (UDS). Results reported here further suggest that NER deficient XP cell lines are sensitive to both ATR and CHK1 kinase inhibition, and when combined with nucleoside analogues, this inhibition increases cytotoxicity compared to wild type controls. Furthermore, the requirement of the Mre11 nuclease in the resistance to nucleoside analogues, as well as the topoisomerase I poison, camptothecin was also studied. Topoisomerases that are responsible for resolving topological stress within the genome during replication and transcription are targets for chemotherapeutic attack in cancers by camptothecin and etoposide. Work here builds on published data from S .pombe which showed that the nuclease activity of Mre11 is required in camptothecin resistance in higher eukaryotes (Hartsuiker et al., 2009). Overall this work indicates a potential role of NER factors in repair and stabilisation of nucleoside analogue induced stalled replication forks, that has a dependence on the ATR-CHK1-Cdc25A S-phase checkpoint cascade. Ultimately this may reveal several potential targets for increasing gemcitabine and nucleoside analogue efficacy in cancers which carry NER factor mutations

Item Type: Thesis (PhD)
Subjects: Degree Thesis
Departments: College of Natural Sciences > School of Biological Sciences
Degree Thesis
Date Deposited: 22 Apr 2016 09:26
Last Modified: 18 May 2016 08:47
URI: http://e.bangor.ac.uk/id/eprint/6552
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