Ultraviolet radiation induces the expression of specific genes that increase repair capacity or damage tolerance. The overall goal of this proposal is to understand how induction of damage-responsive genes is regulated using the PHR1 gene of S. cerevisiae as a model system. PHR1 encodes the apoenzyme of a cyclobutane pyrimidine dimer photolyase. The 5 -regulatory region of PHR1 contains three cis-acting elements that contribute to basal level and induced transcription: an Upstream Repressing Sequence that binds the damage-responsive repressor Prp, an Upstream Activation Sequence (UAS) that is required for basal level and induced expression, and an Upstream Essential Sequence that antagonizes repressor function. The researchers will identify proteins that act through these elements and elucidate the role of each in the regulation of PHR1 expression and expression of other damage responsive genes. They have identified an ORF of previously unknown function which they believe encodes the DNA binding component of Prp. The role of the encoded protein (Orf-p) in gene expression and Prp function will be addressed using temperature-sensitive mutants and strains that overproduce Orf-p. Immunological approaches will be used to directly demonstrate the presence of Orf-p in the Prp complex. The binding site preference of Orf-p will be elucidated with the goal of identifying other binding sites in the yeast genome. Mechanisms controlling Orf-p activity following DNA damage will be investigated. Genetic screens will be performed to identify regulators of UAS activity. The genes will be cloned and the encoded proteins tested for DNA binding in vitro and transactivating activity in vivo. DNA binding studies and mutant analysis will be used to test the role of these proteins in controlling other damage-responsive genes. The close proximity of the cis-acting elements in the PHR1 promoter suggests that proteins bound at these sites interact with one another. Evidence will be sought using the yeast two-hybrid system. Two hybrid screens will be performed to identify other interacting proteins including components of signal transduction pathways that impinge upon these regulatory proteins and control their activity in response to DNA damage. Numerous transcriptional regulators and signal transduction pathways are conserved between yeast and humans. Thus, the results of these studies are likely to yield insight into the regulation of the damage response in humans, and in particular to mechanisms underlying carcinogenesis.
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