Xeroderma pigmentosum (XP) and Crockayne's syndrome (CS) are human disorders that are characterized by increased sensitivity to UV light and a range of developmental and neurological abnormalities. In addition, XP patients show a high predisposition to skin cancer. Cells from patients with both diseases are defective in nucleotide excision repair (NER), and fall into multiple complementation groups that represent different genes of the NER pathway. Additional work has demonstrated that the products of some of these NER genes have dual roles as components of the transcription factor TFIIH. The involvement of these gene products in transcription begins to explain the pleiotropic effects of the XP mutations. However, even though these proteins play a direct role in transcription, the abnormalities associated with XP are generally limited. This suggests that these proteins may be involved in specific developmental decisions. These important developmental processes remain to be identified. We propose to use the multicellular slime mold, Dictyostelium discoideum to study the specific developmental and cellular roles of XP and CS related genes. Although relatively simple, Dictyostelium shares many of the developmental processes of higher eucaryotes. Its ease of study has resulted in a detailed picture of its development at both the cellular and molecular levels. Molecular biology techniques, including those for homologous gene disruption, are well developed allowing facile construction and study of specific mutants. Phenotypes can be studied in detail using a wide range of existing techniques, probes and antibodies. We have identified and studied the Dictyostelium repE gene which encodes a predicted product that is homologous to the human UV-DDB (UV-damaged DNA binding) protein, The UV-DDB protein is absent from some patients with XPE. The predicted repE protein encodes a putative leucine-zipper motif and an associated basic region. We have made repE gene disruption mutants, and they have a unique defect in development. The mutant cells are blocked in aggregation and formation of multicellular assemblies. Interestingly, the mutant cells can express all the early developmental genes tested and acquire the cellular morphology of normal aggregating cells. However, late developmental genes are not expressed. These data suggest that the repE gene acts as a specific developmental switch linking early and late development. We propose aims to analyze specific aspects of this developmental role including; 1) the developmental regulation of repE; 2) the protein and DNA interactions of the repE gene product; and 3) the biochemical and cell biological defects responsible for the phenotype of the repE mutants. We have also identified the Dictyostelium homologs of the XPB and XPD genes which have a variety of developmental defects in humans. We propose to clone and disrupt these genes so that their developmental roles can be studied. Overall, our data indicate that Dictyostelium is useful in elucidating the specific developmental and cellular roles of XP and CS genes.
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