The long-term objective of this project is to understand the molecular basis of DNA repair deficiency in xeroderma pigmentosum (XP). The overall experimental strategy is to simplify the analysis by using agents such as UV that produce only a few types of lesions, using specific assays for each of these lesions, and using defined DNA sequences as substrates for repair so that the processing of damage can be analyzed at the molecular level. The following three experimental approaches and specific aims are planned: (1) We have discovered that XP cells from complementation group C exhibit an intracellular heterogeneity in the repair of DNA containing pyrimidine dimers. Some regions of the genome are repaired normally while others are relatively inaccessible to repair. We plan to analyze this response to determine what distinguishes repairable from non-repairable regions, using the methodology we have developed to isolate the repaired """"""""domains."""""""" We will determine whether such heterogeneity is also a characteristic of other XP complementation groups. (2) Having obtained the first monoclonal antibody specific for thymine glycol in DNA we will use it to study the repair and relative biological significance of thymine glycol in normal and XP cells. We plan to prepare additional monoclonal antibodies and to use them as highly sensitive, specific assays for pyrimidine dimers and other well-defined photoproducts. Results obtained from immunological assays will be compared to those obtained using specific endonucleases to detect lesions and their distribution in the genome. (3) We will use defined chimeric plasmids to probe specific features of DNA repair in XP cells. We have found that 254 nm UV doses below 200 J/m squared enhance the transformation efficiency of pSV2-gpt while doses above 1000 J/m squared cause a decrease which is more pronounced in XP-A than in normal cells. We will determine the nature of the inactivating lesions and the cellular events that underly reactivation in normal cells. This research should contribute substantially to our understanding of the basis for DNA repair deficiency in XP and will also result in availability of new, sensitive probes for the analysis of damage and repair in human cells.
