The highly conserved Saccharomyces cerevisiae Rad23 protein was originally identified as a DNA repair factor and has several homologues, including the human hHR23A and hHR23B proteins. Early on sequence analysis suggested a role for Rad23 in the ubiquitin/ proteasome system (UPS), which is responsible for the degradation of redundant proteins in the cell. Rad23 has been shown to be involved in regulation of nucleotide excision repair (NER), proteolysis and cell cycle progression. The precise function of Rad2 3 is unknown, increasing evidence suggests a close interplay between Rad23 and the UPS is essential for its diverse functions. In order to study the role of hHR23B/Rad23 in NER, I will generate mammalian cell lines and yeast strains that stably express re spectively hHR23B or Rad23 and various mutants (lacking various domains or ubiquitination sites) tagged with green fluorescent protein (GFP). Different fluorescence-based procedures will be applied to study protein kinetics and behavior in living cells. T o further dissect the biological importance of hHR23B/Rad23 ubiquitination I will perform two genome-wide high-throughput screens in yeast to look for proteins, which are involved in the ubiquitination of hHR23B/Rad23. I will also determine the possible role of these proteins in NER.
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