The long term objectives are to define the different biological functions of genes required for excision repair of ultraviolet (UV) damaged DNA in the eukaryote, Saccharomyces cerevisiae, determine the biochemical activities of their protein products, and reconstitute the damage specific incision activity. This proposal focuses on five excision repair genes: RAD1, RAD2, RAD3, RAD4, and RAD10, and their encoded proteins. Studies will be undertaken to define the role of RAD3 in excision repair and viability. The effect of superhelicity, ATP binding vs. its hydrolysis, and other factors on specific binding of RAD3 protein to UV damaged DNA will be determined, and the role of RAD3 in stimulating strand displacement synthesis by yeast DNA polymerase examined. The possible involvement of RAD3 in coupling excision repair and transcription will be investigated by examining whether the rad3 Arg48 mutation defective in helicase activities, and cs rad3 mutations (identical to mutations in the human XPDC gene, the RAD3 homolog, that cause Cockayne syndrome, CS), are defective in preferential repair of the transcribed DNA strand. The rad3 ts14 mutation that stops growth rapidly at 37oC will be further analyzed for its effects on transcription and DNA replication. The possibility that the severe transcriptional defect observed in the rad3 ts14 mutant at 37oC arises from a defect in RNA chain elongation or in promoter specific transcription initiation by the three RNA polymerases will be tested. The interaction of RAD3 with RNA polymerase II and the TATA binding factor will be examined by co- immunoprecipitation and protein-affinity chromatography. Genes encoding proteins that interact with RAD3 will be identified using the genetic strategy of the two hybrid system. Interactions among purified RAD proteins will be examined by hydrodynamic studies, protein affinity chromatography, and other methods. The stoichiometry and other biophysical properties of the RAD1/RAD10 complex will be determined. The role of the leucine zipper motif in RAD1`, and of two contiguous helix motifs in RAD10, in complex formation between these two proteins will be ascertained. Purified RAD1 protein will be examined for its biochemical activities alone and in combination with purified RAD10 protein. The RAD2 and RAD4 proteins will be purified and their biochemical activities characterized. Biochemical activities of combinations of RAD proteins will be determined and the enzyme activity for incision of UV damaged DNA reconstituted in vitro. Xeroderma pigmentosum (XP) patients are defective in excision repair of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Because remarkable evolutionary conservation exists among the excision repair genes in eukaryotes from yeast to human, information from the proposed studies with the yeast genes and proteins should serve as a useful model for delineating the biological roles of human excision repair genes, in characterizing the biochemical activities of their protein products, and in defining the incision mechanism in humans.
Showing the most recent 10 out of 48 publications
