Recent studies by others have shown that the
endonuclease complex coded for by the uvrA, uvrB and uvrC genes of Escherichia coli (UVR ABC excision nuclease) can incise
DNA containing a variety of 'bulk-type' lesions, such as those resulting from u.v. light, (+/-)-7 alpha,8 beta-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]
pyrene (anti-
BPDE), and
N-acetoxy-2-acetylaminofluorene. Using partially purified UVR ABC excision nuclease, we have quantitated the number of
endonuclease sensitive sites (ESS) in purified
DNA isolated from human fibroblasts treated with u.v. light or
BPDE. The number of ESS/10(8) daltons of
DNA were calculated from the number average mol. wt. of the
DNA as determined by sedimentation in alkaline
sucrose gradients. 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) had 20-25% more ESS at each dose than the
BPDE-treated normal (HSBP) cells. Cells treated with 4 microM
BPDE and allowed 12 h of incubation to perform excision repair showed removal of 60% of the initial number of ESS from HSBP
DNA and 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 at 48 h only 22% of the initial ESS remained. In cells treated with 10 J/m2 of u.v. light, the UVR ABC excision nuclease detected 60% of the sites recognized by the pyridimine dimer specific Micrococcus luteus glycosylase/apyrimidinic
endonuclease. 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.