Biochemical and genetic analysis of excision DNA repair

grantor: University of TorontoThe integrity of the genetic material is constantly threatened by various DNA damaging agents. Hence, multiple DNA repair pathways have evolved to eliminate damage of different origins. Nucleotide excision repair (NER) is the primary mechanism by which both 'S. cerevisiae' and human cells remove lesions, including those induced by ultraviolet light, that usually cause severe distortions of the DNA helix. I have described a simple protocol for preparing yeast whole cell extracts that can support efficient NER to facilitate biochemical studies of this repair pathway in yeast. This assay reflects ' bona fide' NER as it depends on 'RAD' genes such as ' RAD14' and 'RAD2', which are known to be essential for excision repair. Interestingly, the assay was also dependent on the ' RAD7' and 'RAD16' genes, whose precise roles in NER are uncertain. Using this system, I also demonstrated that yeast replication protein A (Rpa), encoded by the 'rfa'2 gene, is required for NER, since 'rfa2' mutations which confer enhanced UV sensitivity ' in vivo' also resulted in a deficiency in NER 'in vitro'. NER can be divided into two subpathways: global genome repair, which is involved in the repair of both active and inactive genes, and transcription-coupled repair. In the latter subpathway, lesions in the transcribed strands of active genes are more rapidly repaired, but contributions of the transcription machinery to this repair pathway are poorly understood. To determine whether the RNA polymerase elongation factor SII plays a role in excision repair, I examined the effect of deleting the SII gene of 'Saccharomyces cerevisiae'. Lack of SII activity enhanced UV sensitivity, but only in the absence of ' RAD7/16'-dependent global genome repair. This increased UV sensitivity was also seen with RNA polymerase II mutants defective in their response to SII. UV sensitivity was also conferred by other RNA polymerase II mutations that do not affect SII function. Another pathway of excision repair, base excision repair (BER), involves DNA glycosylases that recognize and excise specific types of base damage in DNA, including uracil and thymine glycol. Human uracil DNA glycosylase (UDG) was recently found to interact with the 34 kDa subunit of human RPA. To investigate whether RPA plays a role in BER in yeast and human cells, I set up ' in vitro' BER systems using yeast and HeLa cell extracts. Both of these systems appear capable of performing efficient BER in the presence of several types of lesions. I fractionated HeLa extracts to deplete endogenous RPA and I will test the ability of these fractions to perform BER. (Abstract shortened by UMI.)Ph.D