ChlR1 is a DNA helicase implicated in diverse cellular processes including sister chromatid cohesion and DNA replication and repair. However, the mechanism by which ChlR1 participates in these processes is unknown. Data presented in this thesis show that siRNA-mediated depletion of ChlR1 causes increased sensitivity to chemically-induced replication stress. Treatment of ChlR1-depleted cells with hydroxyurea results in increased mono-ubiquitination of PCNA and increased chromatin-associated RPA, indicating stalled DNA replication. Furthermore, ChlR1 is recruited to chromatin following hydroxyurea treatment, supporting a role in the stabilisation of forks during replication stress. Fibroblasts derived from a Warsaw Breakage Syndrome (WABS) patient caused by mutation of ChlR1 (G57R) have both defective sister chromatid cohesion and G2 checkpoint following radiation-induced damage. Complementation with wild-type ChlR1 rescued this mutant phenotype while a known helicase dead mutant of ChlR1 (K50R) or the WABS-associated mutants G57R or ΔK897 did not. However, increased and prolonged Chk1 activation was observed in both K50R and ΔK897 complemented cells after treatment with hydroxyurea while the G57R was comparable to wild-type. These data suggest that the novel WABS mutation (G57R) may retain some wild-type ChlR1 activity and offer important insight into the molecular basis of the WABS phenotype.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659143 |
| Date | January 2015 |
| Creators | McFarlane-Majeed, Laura |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/5919/ |
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