GENE PRODUCT RELATED TO XERODERMA PIGMENTOSUM GROUP

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The human DNA repair mechanism is a complex system and an extremely important safeguard against DNA damage caused by both chemical and physical agents. A disturbance in the initial step of the excision repair pathway is involved in the human genetic disorder xeroderma pigmentosum complementation group A (XP-A). Phenotypically, this disease is associated with a very high incidence of skin cancer due to a failure of repair DNA damaged by ultraviolet irradiation as well as exposure to carcinogenic chemicals. Identification of the gene(s) responsible for DNA repair is not only important to elucidate the molecular defect of xeroderma pigmentosum, but is also crucial to understanding the mechanism of carcinogenesis in humans. A non-functional cDNA and its related functional genomic cosmid clone (partially complements the XP-A phenotype in transformed affected cells) have been isolated and characterized (Rinaldy et at., 1988, 1990; Mori et al., 1992; Kaur et al., 1992) . The non-functional CDNA and the functional cosmid genomic clone represent a novel gene that requires in- depth characterization in order to determine its role in DNA repair. A fragment of this gene was amplified by PCR and used as a marker for both the repair proficient cells and the partially complemented XP-A cells resulting from transformation with the cosmid clone. It has been demonstrated that this gene is strongly inducible with ultraviolet irradiation and exposure to benzpyrene diol epoxide (BPDE). It is therefore feasible to isolate a functional CDNA clone from the partially complemented XPA cells. The objective of the proposed experiments is to further investigate the gene product derived from the functional cDNA and its association with the defect in XPA. A cDNA library will be constructed from the BPDE induced XPA transformant and screened with the PCR amplified region of the non-functional cDNA. The cDNA will be completely sequenced and expressed using a shuttle vector in the XPA cell line in order to permanently complement the DNA repair defect. In-vitro transcription, followed by micro-injection of the transcript into the XPA cell line will be performed to determine the transient repair capability of the micro- injected cells. Additionally, cDNA will be expressed in E. coli and the gene product will be purified for antibody production. Western analyses will then be employed to detect the level of the gene product in repair deficient XPA cell lines compared to repair proficient cell lines. The translation of the cDNA will also provide valuable primary structural information about the XP complementation group A gene(s).