746 Detection of the small, excised, damage-containing DNA oligonucleotide (sedDNA) products of nucleotide excision repair in the epidermis of UVB-irradiated human skin

      The nucleotide excision repair (NER) system removes pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] and cyclobutane pyrimidine dimers (CPDs) from DNA in the form of small (∼30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). However, the detection of UV photoproduct-containing sedDNAs as a biochemical read-out for NER activity has thus far been limited to defined in vitro cell-free systems and to cultured cells. Using small punch biopsies of human skin, we show here that the sedDNA products of NER can be isolated, detected, and quantified in skin epidermis within minutes of UVB exposure and following low, sub-erythemal doses of UVB. Interestingly, a partially degraded form of the sedDNAs can be detected in human skin epidermis for several hours following UVB irradiation, which indicates that sedDNA degradation occurs slowly following their release from epidermal genomic DNA. Treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB (xeroderma pigmentosum group B), inhibits sedDNA generation following UVB exposure. Our results indicate that the detection of sedDNAs offers a sensitive and quantitative assay for nucleotide excision repair activity in human skin samples and may therefore be useful for determining how genetic, environmental, and pharmacological factors contribute to inter-individual differences in NER activity in human skin.