Defects in the nucleotide excision repair pathway 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 in human and rodent cells. This study initiates biochemical studies of the structure and function of the mammalian nucleotide excision repair protein CXPD, the Chinese hamster homolog of the human XP group D gene (ERCC2).
The specific aims are to elucidate the multiple functions of this protein in DNA metabolism and to identify its possible specialized role in transcription- coupled repair (in addition to its role in overall DNA repair). Understanding these roles will provide insight into the processes of DNA repair and metabolism, vital cellular processes for maintaining genome integrity. The CXPD protein has distinct roles in DNA repair and cell viability and may also have a role in replication. The human and yeast homologs of CXPD are known to have helicase, ATPase, and DNA-binding activities. Characterization of four highly UV-sensitive CXPD mutant cell lines has revealed heterogeneity in the level of removal of (6-4)photoproducts, suggesting a specific role for CXPD in the preferential repair of damage in actively transcribed sequences as well as its role in overall repair. In order to study the biochemical properties of these mutant CXPD proteins, the CXPD cDNA will be cloned into a bacterial over-expression vector. Proteins with the same alterations found in the CXPD mutant cell lines will be produced by site-directed mutagenesis of the cDNA clone. Wild-type and mutant proteins will be purified, characterized both biochemically and enzymatically, and tested for repair capacity in both cellular and cell-free assays. Relating the biochemical activities and the repair capacities of the mutant proteins to the molecular defects and cellular phenotypes will provide insights into the functional domains of CXPD that are necessary for its various roles in DNA metabolism. Frame-shifts are usually a lethal event in a gene essential for cell viability. Functional domains required for repair but not for viability will be identified by determining the CXPD mutations in four highly UV- sensitive cell lines that were induced using a frame-shifts agent. CXPD mutants with putative defects in the replication function will be characterized (construction is in progress). Defects in the replication function should result in increased levels of recombination and mutation. If these mutants display the hypothesized phenotype, this study will provide direct evidence for the replication function in mammalian cells and the corresponding mutant proteins will be purified from over- expression constructs and characterized biochemically and enzymatically. The generation of mammalian hyper-recombination mutants in Chinese hamster ovary cells will provide a valuable tool for future studies into this important process that plays a critical role in mutagenesis, carcinogenesis, and aging.
