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Title: Nucleotide excision repair in the Saccharomyces cerevisiae MFA2 gene
Author: Teng, Y.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 2000
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An end-labelling strategy for mapping DNA damage in yeast Saccharomyces cerevisiae at the level of the nucleotide is presented. Compared with other high resolution damage mapping protocols, the method described in this thesis is simpler and more efficient with less radioactive manipulation. The MFA2 gene, which is actively transcribed in a-mating type cells whereas repressed in α-mating type cells, provides an ideal model system for studying DNA repair due to its small size and easy control of its expression. The data from nucleotide excision repair competent strains showed that repair of Cyclobutane Pyrimidine Dimers (CPDs) in the upstream region of the MFA2 gene is enhanced in the Mcm1 binding region, and the closer to the transcription start point, the quicker the repair rate observed except for the TATA box region. This enhanced repair rate becomes fastest in the MFA2 transcribed region due to the transcription-coupled repair (TCR). The data from the rad16 mutant strain provided the first evidence that Rad16 is not absolutely required to repair CPDs in some upstream control sequences despite being nontranscribed regions. Experiments with a rad26 mutant showed that Rad26 is essential for TCR of the whole MFA2 transcribed region. Here, although TCR was abolished in the transcribed region, enhanced repair still occurred in the control region of MFA2. Studies with yeast replication protein A mutants indicated that the DNA binding domain, not the protein interaction domain of RPA70, confers a defect in global repair and only partly impairs the process of TCR. The experiments with transcription adaptor mutants, Δgcn5 and Δada2, identified a requirement for these gene products in Nucleotide Excision Repair (NER) of CPDs. Comparing the two adaptors examined, Gcn5 plays a more important role than Ada2 in both MFA2 transcription and CPD repair.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available