Characterisation of DNA damage inducible responses and repair in human cells using recombinant adenovirus vectors

The DNA damage induced by UV and a number of chemical agents is repaired by the nucleotide excision repair (NER) pathway. NER itself can be further subdivided into two pathways: global genome repair (GGR) acts to repair damaged DNA equally throughout the entire genome, while transcription-coupled repair (TCR) specifically targets repair activity to transcription-blocking lesions. The genetic disorders Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are characterised by deficiencies in one or both of the NER subpathways.; In order to examine DNA repair processes in human cells, we have taken advantage of recombinant adenovirus (Ad) vectors. We have used recombinant Ad vectors expressing DNA repair genes, tumor antigens and genes implicated in the cellular response to UV. Additionally, repair of damaged Ad DNA has been used as a means of assaying cellular repair capacity. Quantitation of reporter activity in cells infected with UV-irradiated preparations of the Ad-reporter construct provides a rapid and simple means for measuring repair activity in primary human cells. Analysis of repair capacity (using this and other assays) in XP and CS cells expressing the T4 bacteriophage repair gene, endonuclease V, has provided insight into the nature of the repair defects in these cells.; Reactivation of reporter activity from UV-irradiated Ad-reporter constructs has also been compared in UV-irradiated and non-irradiated cells. Moderate UV exposures were observed to increase the capacity of cells from normal individuals to reactivate reporter activity. Examination of this phenomenon in XP and CS cells suggests that it is dependent on viable TCR.; The effect of cellular exposure to DNA damaging agents on expression from reporter constructs driven by the human and murine cytomegalovirus (CMV) immediate early promoters was also examined. Treatment of cells with either UV or chemical DNA damaging agents induced activity from these reporter constructs. Examination of XP and CS cells, and in cells previously infected with a second Ad vector carrying high amounts of DNA damage, indicate that persistent damage in active genes plays a significant role in this response. SV40-transformed cells did not exhibit increased reporter activity in response to UV exposure, and preliminary experiments suggest a role for members of the pRb family, but not p53, in mediating this response.; The results presented provide evidence for a UV-inducible DNA repair and/or damage tolerance mechanism that is dependent on TCR and requires functional p53 activity; and for a cellular response which enhances expression from CMV promoters in response to unrepaired damage in active genes. Additionally, the data herein provide support for additional functions ofthe XP groups F and G, and CS proteins in DNA repair processes. (Abstract shortened by UMI.)...