These studies will utilize the yeast Saccharomyces cerevisiae as a model to investigate the molecular mechanism of nucleotide excision repair (NER) of DNA in eukaryotic cells. Since yeast NER genes are conserved in all eukaryotes thus far investigated, including mammalian cells, these studies are expected to provide considerable information on the molecular basis of the human cancer-prone, hereditary repair-defective diseases xeroderma pigmentosum (XP) and the disease Cockayne syndrome (CS) which is believed to be defective in a transcription-dependent NER mode. Multiple cloned genes required for or involved in NER are now available, as are multiple cloned genes that participate in both NER and RNA polymerase II transcription. Recent evidence suggests that the Rad proteins required for NER as well as the subunits of TFIIH are preassembled into a large NER machine that has been designated as the nucleotide excision repairosome. It is proposed to characterize the structure and function of this multi-protein complex in greater detail and to establish a fully reconstituted NER system in vitro. It is also proposed to determine whether other transcription factors participate in NER and to establish the role(s) of DNA replication accessory proteins such as RFA, RFC and PCNA in NER in yeast. The NER mode which operates in the in vitro system is independent of RNA polymerase II transcription. There is evidence that the yeast RAD7 and RAD16 genes are required for this process. Their role appears to be one of modulating chromatin structure to provide access of the repairosome to sites of base damage. It is proposed to isolate and characterize a putative Rad7/Rad16 chromatin-modulating complex. In addition to transcription-independent NER there is a particular interest in exploring the possibility that a transcription-dependent NER mode also operates in yeast. At least two candidate genes for this process designated RAD26 and RAD28 [the yeast homologues of the human Cockayne syndrome group A (CSA) and group B (CSB) genes, respectively] have been identified. There is reason to believe that the proteins encoded by these genes operate in a different chromatin-modulating complex, and it is proposed to isolate and characterize this putative complex as well.
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