One of the proteins that lie at the heart of the DNA Damage Response (DDR) is Topoisomerase II-binding protein I (TopBP1). TopBP1 was initially identified and has been extensively studied in the yeast model organisms. However, the lack of readily available tools, including genetically defined mutant cell lines, has rendered the characterisation of TopBP1 in higher eukaryotes more challenging. Sequence information obtained from the characterisation of the gallus gallus TopBP1 mRNA revealed a different splicing pattern at the 5'end to the one reported in the Genome Browser. Our assembled TopBP1 mRNA sequence containing a novel open reading frame (ORF) enabled the creation of a conditional knockout cell line of TopBP1 in DT40, which has been impossible with the use of the annotated cDNA sequence. Thus the avian TopBP1 ORF identified herein contained the necessary function(s) to sustain viability of DT40 cells in the absence of the endogenous protein. Additionally, the establishment of an isogenic set of stable cell lines from the chicken B cell line DT40 by targeted deletion of the TopBP1 alleles revealed a gene dosage-dependent reduction of the TopBP1 protein levels and functions. This work establishes a novel gene-dosage system that can be used for the knock in of point mutations within the endogenous TopBP1 locus. Using this system, a novel characterisation of knock-in point mutants of the ATR Activation Domain (AAD) of TopBP1 was carried out, providing in vivo evidence of its DDR function(s). Finally, a stably integrated overexpression system (SIOS) capable of producing increased amounts of a protein of interest has been established in DT40 cells. SIOS represents an easy to use versatile system for various experimental purposes in the field of DT40. The work presented in this thesis represents a novel characterisation of the avian TopBP1 mRNA and the TopBP1 protein and its functions. This is crucial to gain insight into the mechanistics of the DDR network and the genetic instability characterising cancer development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:731223 |
| Date | January 2017 |
| Creators | Skouteri, Meliti |
| Publisher | University of Sussex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://sro.sussex.ac.uk/id/eprint/71339/ |
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