Bryntesson, Fredrik Anders; (2001) An investigation of genes involved in doubled-stranded break repair of DNA. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Damage to DNA can lead to genomic instability and therefore poses a great threat to the living organism. Living organisms have therefore evolved responses to DNA damage that help maintain genetic stability. These responses include DNA repair mechanisms. DNA damage that leads to double-stranded breaks of DNA is especially threatening. In mammals the main pathway to repair double-stranded breaks of DNA is non-homologous end joining (NHEJ). The proteins Ku and DNA-PKcs that form the DNA-dependent protein kinase (DNA-PK) are critical components of NHEJ, but their exact roles in this process remain unclear. A number of reports have suggested that Ku and DNA-PKcs act as modulators of transcription, but comprehensive in vivo evidence to support this hypothesis is lacking. To directly address whether Ku or DNA-PK regulates gene transcription following DNA damage this project has employed the PCR-coupled subtractive method of cDNA representation difference analysis. Reciprocal subtractions have been carried out using both established DNA-PK defective cell lines, and primary mouse embryonic fibroblasts from mice carrying a targeted disruption in the DNA-PKcs gene. Differential transcription was detected and confirmed in these subtractions. Evidence is presented that transcription of the LAMA4 gene is regulated by DNA-PK in an irradiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of transcriptional differences were not consistently dependent on DNA-PK status. Finally, in a collaboration project, it is demonstrated that DNA-PK is not required for the p53 dependent response to DNA damage. These results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

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