Several skin diseases are caused by alterations in the normal resistance of the skin to sunlight. In one type exemplified by Xeroderma Pigmentosum (XP), these alterations occur because of a decrease in the capacity of cells to repair or replicate the sunlight-induced DNA damage. For many of the XP complementation groups, their associated genes have been cloned, and the functional analysis of those proteins are currently underway. However, in roughly 20% of XP patients designated as """"""""XP variants"""""""" (XPV), the nature of the DNA repair and/or replication defect remains a mystery. To target a selected gene, we have taken advantage of oligonucleotides which bind to duplex DNA and form triple helices (triplex DNA) in a sequence specific manner. One mutagen, psoralen, is linked to the oligonucleotide at its 5' end. Using the pSP189 shuttle vector containing the supF gene as a mutation reporter gene, we challenged the XPV cells with triplex-targeted psoralen crosslinks and monoadducts and analyzed the resulting mutations. Our results show that the psoralen crosslinks are highly mutagenic in an unusual way in XPV cells. The pattern of mutations from both UVA and visible light indicates that XPV is distinctly different from normal or other XP cells. We propose to use this shuttle vector assay to examine the mutagenesis and recombination pathways in XPV cells. Using this same assay we will also try to evaluate the most likely candidate genes, (e.g. human pol beta, pol delta, pol epsilon, RP-A or PCNA) that might alter the XPV phenotype. The standard gene transfer techniques will be the tools for these experiments. Since the recombination step has been proposed as important for bypassing interstrand crosslinks, we will use our assay system to investigate the recombination pathway in XPV cells. All these experiments are based on our preliminary results, suggesting that there is a defect in the processing of triplex-targeted psoralen adducts in XPV cells. Our long-term goal is to elucidate the pathways in XPV cells in order to understand the pathogenesis of the disease. Our plan would be to use the results from these pilot studies to seek additional funds to expand the research program.
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