The long-term objectives are to understand how the Saccharomyces cerevisiae RAD6 protein, a ubiquitin conjugating (E2) enzyme, functions in DNA repair, UV mutagenesis, and sporulation; to purify and characterize the biochemical activities of the RAD18 and REV2 proteins involved in error free postreplication repair, and to study the RAD6, REV1, and REV3 proteins required for UV mutagenesis.
The specific aims are to isolate rad6 mutations that affect only the repair, mutagenesis, or sporulation functions and to obtain and characterize multicopy suppressors of such rad6 mutations. It is expected that multicopy suppressors will identify genes that encode proteins interacting with RAD6. RAD6 polyubiquitinates histones H2A and H2B in vitro and the acidic carboxyl terminal region of RAD6 protein is required both for histone polyubiquitination and for sporulation. The in vivo role of RAD6 in histone ubiquitination will be examined by determining the level of histone ubiquitination in wild type RAD6/RAD6 and mutant rad6/rad6 diploids during sporulation and in RAD6 and rad6delta cells after UV irradiation. The role of RAD6 mediated E3 dependent protein degradation will be examined in vegetative and sporulating cells by determining the stability of X-beta gal fusion proteins that differ only in residue X, which can be a stabilizing or destabilizing residue. To determine whether RAD6 is involved in protein degradation during sporulation, protein degradation will be measured in RAD6/RAD6 and rad6delta/rad6delta cells labeled uniformly with 35S- methionine. A gene will be cloned from Schizosaccharomyces pombe that restores sporulation ability to the sporulation defective S. cerevisiae rad6-149/rad6-149 mutant, which lacks the carboxyl terminal 23 predominantly acidic residues. The RAD18 and REV2 proteins will be purified from yeast carrying RAD18 or REV2 overproducing plasmids and the purified proteins examined for DNA binding, ATPase, DNA helicase, strand exchange and other biochemical activities. The cellular localization of the RAD18 and REV2 proteins will be determined by indirect immunofluorescence. Complex formation among the RAD6, RAD18, and REV2 proteins will be examined by co-immunoprecipitation experiments. We will determine whether the S. pombe, Drosophila or human RAD6 homologs can substitute for yeast RAD6 in complex formation with RAD18. We will examine if RAD6, REV1, and REV3 proteins form a complex, and whether the REV1 and REV3 proteins become modified by RAD6 dependent ubiquitination. Phosphorylation of REV1, REV3, and RAD6 proteins by the CDC7 encoded serine(threonine) protein kinase, which is involved in UV mutagenesis, will be examined.
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