Genome wide analysis of dna repair by expression profiling

Ridley, Andrew James. 2006. Genome wide analysis of dna repair by expression profiling. PhD Thesis, Cardiff University.

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Abstract

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are syndromes characterised by defects iqf nucleotide excision repair (NER), they can be distinguished by contrasting clinical manifestations. Although the genes responsible for XP and CShave been identified, the precise molecular roles of the normal proteins remains poorly understood. In the present study, primary dermal fibroblasts derived from patients assigned to XP complementation group C (XP-C XP8CA) and CS type A (CS-A CS3BE) were characterised. Patient XP8CA was homozygous for a 2 bp TG deletion in the XPC gene at codon 547 resulting in a premature termination at position 572, while patient CS3BE was a compound heterozygote for a 37G>T (E13X) and a novel 479C>T (A 160V) mutation in CKNl, the jene that encodes the CSA protein. Permanent XP-C and CS-A cell lines were established by transducing primary XP8CA and CS3BE fibroblasts with a retroviral vector, expressing the catalytic subunit of telomerase, hTERT. The reconstitition of telomerase activity resulted in: (1) the preservation of the primary NER capabilities (2) an extension of proliferative lifespan (3) maintenance of the p53/p21WAF/CIPI and pRb/pl6INK4A tumour suppressor pathways. Using microarrays, the UV-induced global transcriptional response of telomerised XP-C ajd CS-A fibroblasts was characterised. The data indicate that UV-irradiation resulted in the differential regulation of a diverse range of cellular responses such as transcription, cell cycle arrest, DNA repair and apoptosis. Additionally, cell type-specific signatures were observed in telomerised XP-C and CS-A fibroblasts. The utility of RNAi was also demonstrated by transiently ablating XPC of CSA function in telomerised repair competent (MRC-5) fibroblasts, and a stable, permanent mutant was constructed by retrovirally transducing the telomerised CS-A cell line with an PC-specific shRNA construct. Thus, permanent and stable telomerase-immortalised XP-C and CS-A cell lines have been established and partially characterised at both the genetic and molecular level, so providing in vitro models for investigating NER.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > RB Pathology
ISBN:	9781303208379
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Jan 2018 04:49
URI:	https://orca.cardiff.ac.uk/id/eprint/56167

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