GENETICS OF HUMAN DNA REPAIR

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The human hereditary syndrome Xeroderma pigmentosum (XP) results from inborn errors in the genes for DNA excision repair. A strong correlation exists between defective DNA repair and carcinogenesis. XP patients exhibits a 2000-fold increase in the occurrence of epidermal neoplasms and a 10-fold increase in the occurrence of internal neoplasms. The high frequency of epidermal neoplasms is due to an inability to repair DNA damage caused by exposure to sunlight. The elevated prevalence of internal neoplasms is probably linked to the heightened sensitivity of XP cells to dietary carcinogens. Nine complementation groups have been defined in XP. Of all the groups, XP complementation group A (XPA) cells have the lowest DNA repair capability. Like others with extremely low DNA repair capability, XPA patients often have associated neuropathy indicating an essential role for the XPA gene product in neural tissue. The objectives of the current proposal are aimed at gaining an understanding of the mechanisms of DNA excision repair. Transformed XPA fibroblasts which have been complemented by DNA mediated gene transfer of a human fibroblast cDNA expression library have been obtained in this laboratory. The plasmid containing the complementing cDNA will be isolated from the genomic DNA of the transformed XPA cells by plasmid rescue. The nucleotide sequence of the cDNA will be determined and the sequence of the encoded protein will be inferred from the cDNA sequence. The protein and nucleic acid sequence data banks will be searched for proteins homologous to the XPA protein in an effort to identify the function of the XPA gene product. The cDNA will be used as a probe to define the molecular defects in XPA cells. This will yield information on regions of the XPA gene product which are critical to its function. XPA specific mRNA expressed in XPA cells will be examined for quantitative or qualitative changes from normal by blot hybridization analyses. Sites of deletions and insertions in the XPA specific mRNAs of XPA cells will be defined by S1 protection mapping. Point mutations affecting protein function will be identified by cloning and sequencing cDNAs to mutant XPA mRNAs. The presence of large deletions and insertions in, and rearrangement of, the XPA specific gene in XPA cells will be identified by blot hybridization analyses of XPA cell genomic DNAs. The normal DNA repair gene will be isolated and characterized. Rodent cells contain an activity which can complement XPA cells. It is unknown whether this activity is the same protein as the XPA gene product. The vertebrate phylogeny of the DNA repair gene will be investigated by blot hybridization analyses of genomic DNAs from various vertebrate organisms with the cDNA probe. The degree of conservation will be estimated by varying the stringency of hybridization with the human cDNA probe. The tissue distribution and levels of expression of the homologous DNA repair gene in mice will ascertained by blot hybridization analyses of RNAs with the human cDNA probe.