Genetic and molecular characterization of Saccharomyces cerevisiaerad25 gene

Cells from patients suffering from xeroderma pigmentosum (XP), a cancer-prone, photosensitivity disorder, show high levels of UV sensitivity. The XPB gene complementing UV sensitivity in the XP-B cell line, XP11BE, (one of the seven complementation groups of XP) had been cloned (Weeda et al., 1990). Genetic and molecular characterization of RAD25, the yeast homolog of the human XPB gene, is the subject of this project. RAD25 was shown to be an essential gene and, when sequenced, revealed a DNA helicase motif. Deletion of the C-terminal 45 amino acids resulted in a UV sensitive phenotype, suggesting that the C terminus is required for DNA repair. The C-terminal deleted rad25 mutant, rad25{dollar}sb{lcub}799am{rcub},{dollar} was used to determine that RAD25, as expected, belongs to the nucleotide excision repair pathway.; In contrast, an altered C terminus resulted in a lethal phenotype, suggesting that specific interactions may be important in performing the essential function. It is clear that the C terminus is not required for essential function since rad25{dollar}sb{lcub}799am{rcub}{dollar} is still viable. Changing the lysine in the nucleotide-binding Walker type A motif (a part of the helicase motif) to arginine also resulted in a lethal phenotype. This suggested that the ATPase or helicase activity predicted by the helicase motif was important for viability.; Random mutations of rad25 were also isolated and used by others in the laboratory to show the involvement of RAD25 in transcription. Three types of mutants were isolated: temperature-sensitive, UV sensitive and several that were both temperature-sensitive and UV-sensitive. Mapping of the mutants showed no regional correlation between the phenotypes and the positions of the mutations.; RAD25 was screened for interaction with other proteins using the two-hybrid system. Two proteins, SPT6, a suppressor of ty insertion expression (Clark-Adams and Winston, 1987), and a novel gene product, were found, that interacted specifically with RAD25 by this method. The novel gene, EP71, was cloned and sequenced and the largest open reading frame encoded a 321 amino acid protein. Genomic deletion of EP71 resulted in neither UV-sensitivity, temperature-sensitivity nor mms (methylmethane sulfonate) sensitivity.