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The construction and employment of a system for the in vivo and in vitro analysis of NER in chromatin.

Johnson, Rebecca Dawn 2010. The construction and employment of a system for the in vivo and in vitro analysis of NER in chromatin. PhD Thesis, Cardiff University.

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Abstract

NER is vital for the integrity of the genome, with defects in NER giving rise to the rare human disorder Xeroderma Pigmentosum. This study has seen the construction of a minichromosome containing MFA2 as a model gene. The TAM plasmid model provides a chromatin environment analogous to that seen at the endogenous MFA2 gene, and the nucleosome positions have been determined along with the repair profile in wild type cells, and in cells lacking the general repressor Tup1p. When TUP1 is deleted, repair of CPDs occurs at a faster rate in both the TS and NTS of the MFA2 promoter. Repair was studied in respect to the HAT Gcn5p, a factor responsible for the H3 acetylation (K9 and K14) of MFA2, a prerequisite for efficient transcription initiation and repair. The TAM model showed diminished repair and a reduction in H3 acetylation at the MFA2 gene when GCN5 was absent, confirming the role for Gcn5p in repair at this gene, regardless of the chromosomal context. Repair was also investigated in the absence of the GG-NER factor Rad16p. Rad16p has been implicated with roles in chromatin remodelling, as well as the UV-induced occupancy of Gcn5p to promote H3 acetylation. In TAM, the absolute requisite for Rad16p in GG-NER was overcome, suggesting the chromatin environment within the plasmid differs from that seen at the endogenous MFA2. The TAM plasmid has provided a tool to study NER both in vivo, as reported here, and in vitro, enabled the biochemistry behind the complex mechanism of NER in chromatin to be realised.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:29
URI: http://orca-mwe.cf.ac.uk/id/eprint/54150

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