Date of Award
Doctor of Philosophy (PhD)
Dr. A.J. Rainbow
UV-induced dimers and bulky chemical adducts in DNA are repaired by nucleotide excision repair (NER). Several lesions repaired by NER including UV dimers pose a persistent block to transcription which in turn promotes apoptosis. A specialization of NER, termed transcription coupled repait (TCR), facilitates removal of transcription-blocking DNA damage by a process which is coupled to transcription by RNA polymerase II (RNAPII). It has been proposed that the presence of RNAPII stalled at a lesion facilitates NER by increasing the affinity of repair enzymes for the site of DNA. However, previous results from our laboratory have demonstrated that host cell reactivation of an ultraviolet (UV)-damanged RNAPII transcribed reporter gene is reduced in TCR proficient but global NER deficient fibroblasts. These results indicate that the RNAPII blocked at the site of a UV-induced lesion is not sufficient to promote the preferential repair of that lesion. It was further demonstrated that UV treatment of these cells increased repair of the reporter gene, strongly suggesting that TCR is inducible. In the present thesis, primary human fibroblast strains with well characterized DNA repair phenotypes were used to demonstrate that heat shock enhanced reactivation of reporter gene activity (HSER) and the capacity of UV-irradiated cells to support adenovirus DNA synthesis (viral capacity), reflect TCR of UC-induced DNA damage. HSER requires repair of the transcribed strand of a reporter gene whereas viral capacity requires repair of genomic DNA. HSER and UV enhanced reactivation (UVER) were: dependent on wildtype p53, disrupted in most tumour cell lines examined and deficient in SV40 transformed fibroblasts. These results were confirmed using the viral capacity assay, indicating that TCR of genomic DNA is inducible through a p53 dependent mechanism. The primary activity ascribed to the p53 tumour suppressor gene is transactivation of genes involved in maintaining genome stability. A key downstream target of p53 is the cyclin dependent kinase inhibitor, p21ᵂᵃᶠˡ. This gene product plays a role in mediating p53 dependent G₁ arrest and it has been suggested to play a role in NER. We hypothesized that UV induced activation of p53 is unlikely to result in efficient transactivation of p53 responsiveness genes because UV induced DNA damage blocks transcription. We report here that UV inhibits p21ᵂᵃᶠˡ expression immediately following irradiation. The efficient recovery of p21ᵂᵃᶠˡ expression and subsequent induction of p21ᵂᵃᶠˡ are dependent on TCR of UV induced DNA damage. The delayed induction of p21ᵂᵃᶠˡ expression following UV irradiation is not consistent with the proposed role of ᵂᵃᶠˡ transactivation in NER. The results presented here predict a dose dependent reduction in the efficiency of G₁ arrest following UV. We addressed the consequences of attenuated G₁ arrest on UV sensitivity by transiently expression SV40 large T antigen, polyoma virus large T antigen, E2F-1 and E2F-4 from recombinant adenovirus (Ad) constructs. Expression of these gene products conferred UV sensitivity on normal diploid fibroblasts. In contrasts, stimulation of the p53 dependent G₁ checkpoint pathway with a similar Ad construct expressing p21ᵂᵃᶠˡ led to increased clonogenic survival following UV irradiation. We propose a model in which UV induced lesions lead to increased p53 activity and p53 stimulated DNA repair but persistent DNA damage prevents expression of p53 responsive gene products in a dose dependent manner. In this way, cells with irreparable DNA damage are likely to be eliminated by apoptosis as a consequence of the inability to express anti-apoptotic genes.
McKay, Bruce Campbell, "The Relationship Between the Repair of Ultraviolet Light Induced DNA Damage in Human Cells and the p53 Tumour Suppressor" (1997). Open Access Dissertations and Theses. Paper 1134.