FINDING THE MOLECULAR DEFECT IN XERODERMA PIGMENTOSUM

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The inherited disease xeroderma pigmentosum (XP) is characterized by severe sensitivity to ultraviolet (UV) radiation and a high incidence of skin cancer. The biochemical defect is in a pathway for the excision repair of DNA damage produced by UV, the antitumor drug cisplatin, and many other agents that produce bulky DNA adducts. This DNA repair pathway involves multiple proteins, since there are at least seven genetic complementation groups, A through G. An early step in repair must involve recognition of the site of DNA damage. Recently, a candidate protein for this function, XPE binding factor (XPE-BF), was identified in this laboratory. It binds to DNA damaged by UV, cisplatin, or denaturation; is absent in XP group E cells; and is expressed at increased levels in cisplatin resistant cells with increased DNA repair. The aims of this proposal are to understand the molecular defect(s) in XP more fully by: 1. Characterization o f XPE-BF. To understand how XPE-BF recognizes damaged DNA, the spectrum of lesions that bind XPE-BF will be defined. To investigate the significance of XPE-BF in cancer treatment, be modulation of XPE-BF levels will be studied in cells with differing metastatic potential, in response to tamoxifen and steroid hormones, and in patients undergoing chemotherapy. 2. Isolation of the gene for XPE-BF. The protein has now been purified in quantities large enough to permit cleavage, peptide sequencing, and the definition of oligonucleotides for screening cDNA. The cDNA and gene will be cloned and characterized to understand how XPE-BF acts as a repair protein. 3 . Proof that XPE-BF is defective in xeroderma pigmentosum. XPE-BF will be tested for its ability to: (a) rescue XP group E cells by microinjection of purified protein, and (b) confer resistance to cisplatin and other anticancer drugs in normal cells. Mutations in XP group E patients will be characterized to define the molecular defect in those individuals. 4 . The study of how XPE-BF participates in DNA repair. Several methods will be used to ideally heterologous proteins that interact with XPE-BF. Such proteins may be defective in other XP groups and will define additional steps in the XP repair pathway. Critical domains in XPE-BF for both DNA binding and protein interactions will be defined. The cloned cDNA will be used to produce both wild type and mutated XPE-BF for testing in an in vitro repair system. The overall goal is to understand the biochemistry of DNA repair at the molecular level. Success in this endeavor will have implications not only for XP patients, but also for understanding mutagenesis and cancer susceptibility in all humans.