Cancer and defective DNA repair are intertwined in the autosomal recessive disease xeroderma pigmentosum (XP). Homozygotes are characterized clinically by a high incidence of skin cancer and biochememically by the defective repair of DNA damaged by ultraviolet radiation. Cell hybridization studies have defined at least eight separate complemention groups, so at least eight gene products are involed.
The aim of this proposal is to identify the molecular defect in XP. Two approaches will be explored, both of which use DNA crosslinked by the antitumor drug cisplatin as a probe for detecting the DNA repair defect in XP. Preliminary work has shown that XP cells exhibit a severe defect in the repair of such a probe. One approach will be to clone the DNA repair genes by the transfection of XP cells with a wild type cDNA expression library. Those cells which have acquired the wild type DNA repair gene will be identified by a two step selection, first by their resistance to ultraviolet radiation and second by their ability to repair marker DNA crosslinked by cisplatin. The complementing cDNA will be recloned from repair competent cells by using SV40 sequences as probes to detect transfected DNA. Once cloned, both the wild type and various XP mutant genes will be analyzed to identify functional domains on the proteins. A parallel approach will be to isolate proteins involved in DNA repair. Cisplation crosslinked DNA will be incubated with cell extracts and then subjected to electrophoresis in a low ionic stength polyacrylamide gel, to identify protein-DNA complexes which are specific for damaged DNA and are present in wild type but not XP cells. The proteins will be purified by chromatographic separation methods, assaying the fractions for binding activity to damaged DNA. Partial protein sequences will direct the synthesis of oligonucleotide probes for screening cDNA libraries. This approach may be especially helpful for cloning genes from XP complementation groups with a less severe phenotype. The long range goal is to understand at a molecular level how the DNA repair system recognizes and removes damaged DNA. This work may provide insight into the relationship between defects in DNA repair and oncogenesis.
