DNA Repair and Mutagenesis Induced by Dimer and Non-Dimer Photoproducts Measured with Plasmid Vectors in Xeroderma Pigmentosum Cells
We have used plasmid vector host cell reactivation assays to measure ultraviolet photoproduct-induced DNA repair and mutagenesis in human cells. Plasmids constructed to permit both replication in bacteria and utilization of human host cell enzymes for expression or replication were used. Plasmids were treated with UV in vitro permitting precise quantification of the extent of damage and selective reversal of dimers by photolyase. The treated plasmids were transfected into xeroderma pigmentosum or repair proficient human cells where repair, expression, mutation, or replication proceeded for 2–3 days and then the plasmids or their gene products were harvested and assayed. Repair was measured by determining the extent of expression of enzyme activity coded for by the damaged plasmid. Repair deficient cells showed less activity than repair proficient cells. Similarly, transformation of indicator bacteria by the harvested plasmid was used to assess the plasmid survival and mutation frequency. DNA sequencing was performed on mutated plasmids recovered. Both cyclobutane dimer and non-dimer photoproducts were found to be poorly repaired and to be mutagenic in xeroderma pigmentosum cells.
KeywordsXeroderma Pigmentosum Xeroderma Pigmentosum Group Plasmid Survival Host Cell Reactivation Thymine Glycol
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