Quantitation of carcinogen-induced DNA damage and repair in human cells with the UVR ABC excision nuclease from Escherichia coli

Using partially purified UVR ABC excision nuclease from Escherichia coli, the number of endonuclease sensitive sites (ESS) in purified DNA isolated from human fibroblasts treated with u.v. light or +- 7α, 8β-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzol(a)pyrene diol-epoxide (BPDE) have been quantitated. The number of endonuclease sites increased linearly with increasing doses of either u.v. light or BPDE. The UVR ABC excision nuclease was able to incise a majority of the BPDE-DNA adducts. Xeroderma pigmentosum fibroblasts, complementation group A (XP12BE) has 20-25% more ESS at each dose than the BPDE-treated normal (HSBP) cells. Cells treated with 4μM BPDE and allowed 12 h of incubation to perform excision repair showed removal of 60% of the initial number of ESS from HSBP DNA amd 40% of the ESS from XP-A DNA. Beyond 12 h XP12BE cells lost no additional ESS while HSBP cells continued to lose ESS, although at a slower rate, until 48 h only 22% of the initial ESS remained. These results demonstrate the potential use of the UVR ABC excision nuclease in a quantitative assay for determining the number of carcinogen-induced lesions in human DNA. (author)