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Title: Investigating DNA Double Strand Breaks (DSB) in mammalian cells by novel fluorescent reporters
Author: Riches, Lucy C.
ISNI:       0000 0000 7210 802X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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An efficient DNA damage response is critical for maintaining the integrity of the mammalian genome, and ensuring the accurate transfer of genetic information between generations. Of particular biological relevance are DNA double strand breaks (DSB), which if repaired incorrectly may contribute to carcinogenesis. Review of contemporary literature has led to the identification of protein interactions and transcriptional events, tightly associated with the mammalian DSB response. Characteristics of selected events have been manipulated, with the notion of developing a reporter system that offers a sensitive and rapid method of detecting DSB in living mammalian cell models. Work presented here provides a quantitative evaluation of DSB generation in various mammalian cell lines, following chemical and irradiation treatment, and highlights the limitations of currently used markers. A series of recombinant proteins comprising peptide interacting domains, which exhibit altered spatio-temporal dynamics in relation with each other following DSB induction, are proposed as potential reporters of damage in mammalian cells. Novel gene constructs have been engineered that encode these peptide interacting domains, sandwiched between fluorescence-resonance-energy transfer (FRET) capable proteins. DSB specific events are predicted to induce peptide interactions that may be tracked in real time, by monitoring alterations in the fluorescent properties of such a recombinant protein. In an alternative approach, the transcriptional up-regulation of RAD52 mRNA following DSB induction was extended to whole cells. Optimisation of a fluorescent molecular beacon probe complementary to mammalian RAD52 mRNA is described, and data obtained in mammalian cells following DSB induction supports the notion that RAD52 is actively transcribed as part of the DSB response.
Supervisor: Gooderham, Nigel ; Lynch, Anthony Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC) ; GlaxoSmithKline (GSK)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral