MOLECULAR CLONING OF HUMAN DNA REPAIR GENES

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A highly efficient mechanism to repair damage to nucleotides in DNA (whether caused by radiation or chemical challenge) is a necessary function of a cell in order to preserve its genetic integrity. A failure to repair these damages or an inaccurate repair mechanism will result in mutations within DNA sequences, which in turn can be associated with direct mutagenesis and ultimately carcinogenesis. There appears to be a strong case for a causal relationship between defective repair of ultraviolet light (UV) damage and carcinogenesis in the human genetic disease, xeroderma pigmentosum (XP). This proposal intends to enhance the understanding of the DNA repair system which is normally responsible for the repair of UV damage in human cells. Novel strategies are proposed for the cloning and expression of one of the genes which is directly associated with the initiation of DNA repair at sites of UV damage in human cells; the deficiency or nonfunctionality of this gene product defines XP complementation group A (XP-A). Relying on the findings of Legerski et al. (1984), that the size class mRNA containing the XP-A gene was 10.6 S, three 10.6 S mRNA cDNA libraries have been constructed: one from an XP-A effected child and two from the obligate XP-A heterozygote parents of the XP-A child. The individual libraries have been used to triplicate screen a fourth human cDNA library in order to identify plaques which are common to both parents but either are not expressed or are overexpressed in the child. Genes which have been identified by these procedures will be further investigated as follows. The gene(s) will be subcloned into a mammalian shuttle vector, transfected into XP-A cells and the cells assayed for enhanced UV survival. The putative DNA repair gene(s) will be used to assay for abundance and any size heterogeneity of mRNAs from other XP-A cells by Northern analyses. In addition, these DNAs will be used to identify restriction fragment length polymorphisms in other XP genomic DNAs. The expression vector Lambdagt11 will be used to produce fusion proteins of Beta-galactosidase and the DNA repair gene product. Antibodies will be raised against the fusion protein and will be used for inhibition of an in vitro thymic dimer excision assay or nicking activities.