Nucleotide excision repair (NER) is the major cellular repair system which operates in human cells to remove a broad spectrum of DNA damage, including photoproducts from UV radiation, oxidative damage from ionizing radiation, and base mono-adducts and cross-links resulting from numerous chemicals. In vivo, NER prevents spontaneous and environmentally induced mutations that can initiate cells on a pathway toward malignancy. A molecular understanding of NER may lead to improvements in cancer prevention and cancer therapy, and also have relevance to other diseases and aging. This long-term goal requires characterization of the participating proteins toward eventual reconstitution of the process. The objective of this proposal is to determine the enzymatic and structural roles of the human ERCC4 NER protein. The knowledge and reagents (proteins and antibodies) obtained will accelerate understanding repair and related DNA metabolism. Our recently cloned ERCC4 gene, one of at least seven genes that are required for NER in human cells, is defined by its ability to correct hamster cell mutants in complementation group 4. The availability of cDNA clones provides the basis for overproducing the ERCC4 protein for the specific aims of functional studies of its precise biochemical role in repair and recombination. By using affinity tags and other means, overexpressed ERCC4 protein will be isolated from both bacteria and human cells, extensively purified, and characterized in terms of single-strand specific endonuclease activity, DNA binding, functionality in cell-extract repair assays, as well as interactions with ERCC1 and other proteins such the XPA damage-recognition factor. The clear homology of ERCC4 with S. cerevisiae RAD1 implies that ERCC4 exists in cells in a complex containing ERCC1, and perhaps other proteins, that mediates the endonucleolytic incision step on the 5' side of a damaged site (e.g. thymine dimer). Therefore, complexes of ERCC4 and ERCC1 will be formed with purified proteins and also isolated from human cells. Native ERCC4 complexes will be characterized to identify the individual proteins. Peptide-specific and monoclonal antibodies will be produced as essential reagents for these studies. In addition, to examine the possible enzymatic role of ERCC4 in recombinational repair, the ability of ERCC4 (complex) to remove single- strand regions of heterologous DNA in synthetic, model substrates will be tested. Mutational changes in ERCC4 structure within highly conserved segments, functional motifs, and other regions will be incorporated into overexpression plasmids. These mutations will be correlated with changes in cellular repair capacity, in vitro biochemical activities such as endonuclease, and complex formation.
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