ISOLATION AND ANALYSIS OF HUMAN DNA REPAIR GENES

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Xeroderma pigmentosus (XP) is an autosomal, recessive genetic disease of humans characterized by a high predisposition to cancers of the skin and neurological complications. XP cells are deficient in the incision step of excision repair and the highly elevated risk of skin cancer to radiation exposed areas of the body are attributed to this detect. XP is genetically complex in that nine different complementation groups have been identified. DNA repair genes that complement the deficiency in XP cells have proven refractory to cloning by traditional methods. Recently developed Epstein-Barr virus (EBV) plasmids which replicate extrachromosomally in human cells, have several advantages over existing expression vectors. They transform human cells at high frequency, express cDNA inserts at a consistent and high level, rearrange at low frequency and are easily rescued from stably transformed cells. Isolation of DNA repair genes will be attempted by transfection of EBV-based cDNA expression libraries into XP cells and identification of repair-proficient transformants by phenotypic selection with UV irradiation. Rescue of putative DNA repair clones will be performed by Hirt extraction and recovered into bacterial cells by electroporation. Structural and biochemical analyses of cloned genes will be performed. These studies should contribute to the long term goals of elucidating the molecular mechanisms of human, DNA excision repair and the radiation sensitivity of XP.