Organisms respond to UV radiation and other types of DNA damage by altering the expression of specific genes that increase cellular repair capacity or damage tolerance. The importance of these processes to human health is apparent in the increased cancer frequency seen in individuals suffering from DNA-damage-processing and -response deficiencies such as xeroderma pigmentosum, Li-Fraumeni syndrome, and ataxia telangectasia. The research described in this proposal addresses fundamental mechanisms regulating the transcriptional response to UV-induced DNA damage in the model organism S. cerevisiae. The proposal focuses on the PHR1 gene, which encodes the DNA repair enzyme photolyase. Rph1 and Gis1 have been identified as damage-responsive regulators of PHR1, and evidence is presented for the presence of at least one other component of the repressor. Additional repressor components, as well as proteins that interact with the complex or its components, will be identified using genetic screens and affinity purification techniques. The sites of interaction between repressor components will be identified using similar techniques, and mutants defective in these interactions will be generated to test the roles of these interactions in vivo. Previous work has shown that following DNA damage the repressor complex dissociates from the PHR1 promoter. Proposed experiments will explore the molecular mechanisms that underlie this event. In particular, the role of phosphorylation, cytoplasmic-nuclear shuttling, altered stability and changes in protein- protein interactions will be explored. Yeast genomic arrays will be used to identify the entire repertoire of genes regulated by Rph1 and Gis1 as well as all genes induced in response to UV and to other stresses. Cluster analysis will be used to identify distinct subsets of genes regulated by one or the other repressor and in response to different stresses, and comparison of promoter sequences of similarly regulated genes will be used to identify coregulators. Many transcriptional regulators discovered in yeast have homologues in humans. Thus, it is likely that identification and characterization of damage-responsive transcriptional regulators in yeast will lead to the discovery of similar regulators in humans. Ultimately this knowledge may help us to identify individuals at high risk for sunlight-induced cancers, as well as other forms of cancer.
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