MOLECULAR CLONING OF HUMAN DNA REPAIR GENE(S)

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Maintenance of the genetic material requires a mechanism for repairing damage to the DNA caused by a variety of chemical and physical agents. If DNA repair fails to occur, mutations may arise in the descendants of the affected cells which in turn can be phenotypically associated with neoplastic transformation. A causal relationship between defective repair of ultraviolet light damage to DNA and carcinogenesis is very strong in the disease xeroderma pigmentosum. The objective of this proposal is to clone and characterize the gene coding for the protein which is responsible for the initiation of DNA repair and lacking in xeroderma pigmentosum complementation group A (XP-A). The size class of mRNA containing the XP-A gene in HeLa cells is within the 10.6S pool (Legerski et al., Proc. Natl. Acad. Sci. U.S.A. 81, 5676, 1984). cDNA libraries derived from the pool of this mRNA of an XP-A affected child and the obligate XP-A heterozygous parents have been constructed and used in hybridization competition strategy to screen both the parents' and a normal human adult liver cDNA library. Of the 15 isolated cDNA clones, 14 comprise one gene family. One of these clones has been partially characterized in terms of nucleic acid sequence, the results of which indicate that this is a novel gene. A fragment of this gene was used for 1) Northern blot analysis and 2) TaqI RFLP analyses of the parental and child DNA. The objective of the proposed experiments is to investigate the relationship between this novel gene and the disease state of xeroderma pigementosum. A human cosmid library constructed by using expression cosmid vector pCV108 containing a neomycin- resistance gene marker will be screened. Cosmid clones will be restriction mapped and used for the transformation of the XP-A cell line and selected for both neomycin and UV resistance. XP-A cells which acquire enhanced UV-resistance as a result of cosmid transformation, will be subjected to Southern as well as Northern blot analyses and these results compared with those of a non- transformed XP-A cell line. In addition, this novel gene will be used 1) to study the population genetics of the other XP-A family cell lines by using Northern and RFLP analyses, and 2) for cytogenetic analyses of other XP-A family cell lines in comparison with both aneuploid VA13 repair-proficient cell line (Schultz et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4176, 1987) and normal diploid cell line.