Defects in the nucleotide excision repair pathway, one of several DNA repair systems, are responsible for the series of cancer-prone genetic disorders called xeroderma pigmentosum (XP). The genetic and biochemical complexity of this repair process Is reflected in the existence of multiple complementation groups. The basis for future biochemical studies of the structure and function of the mammalian nucleotide excision repair protein ERCC2 will be provided by this study to elucidate the multiple functions of ERCC22 and identify the role of ERCC2 in the preferential repair of UV-induced DNA adducts. The ERCC2 protein has distinct roles in DNA repair, recombination, and cell viability. ERCC2 mutants with defects in either 'he replication or essential functions will be created by site directed mutagenesis and targeted recombination using CHO cells, made possible since ERCC2 is fortuitously single copy in these cells. Defects in the replication function should result in increased levels of recombination and mutation. This study will provide direct evidence for these functions in mammalian cells. The generation of mammalian hyper-recombination mutants will provide a valuable tool for future studies into this important process that plays a critical role in mutagenesis and carcinogenesis. Characterization of four UV-sensitive hamster ERCC2 mutants has revealed heterogeneity in the level of removal of (6-4)photoproducts, suggesting a role for ERCC2 in the preferential repair of damage in actively transcribed sequences. The specific molecular defect in the ERCC2 gene of these four mutants and the change in three partial revertants will be identified using PCR and direct sequence determination. In order to accomplish the preceding goals, the nucleotide sequence of cDNA clones and genomic intron/exon junctions and flanking regions will be determined. The wild-type hamster ERCC2 clones for these determinations will be isolated using a previously isolated human cDNA probe In order to study the biochemical properties of the mutant ERCC2 proteins, the hamster ERCC2 gene will be cloned into a yeast expression vector and site directed mutagenesis will be used to introduce the same defects as are in the mutants described above. Mutant proteins produced from these clones will be used for biochemical and enzymatic characterization in subsequent experiments. Relating the biochemical activities of the proteins to the molecular defects and cellular phenotypes will provide insights into the functional domains of ERCC2 that are necessary for its various roles in DNA metabolism. Understanding the multiple roles for ERCC2 will provide insight into the processes of DNA repair and metabolism, vital cellular processes for maintaining genome integrity. In addition to furthering our understanding of fundamental aspects of DNA metabolism, this study will provide the foundation for constructing an animal model for studying the relationship of differences in DNA repair capacity to differential susceptibilities to carcinogenesis.
