A P-32-Postlabeling assay for the oxidative DNA lesion 8,5 '-cyclo-2 '-deoxyadenosine in mammalian tissues - Evidence that four type II I-compounds are dinucleotides containing the lesion in the 3 ' nucleotide

8,5 ' -Cyclopurine-2 ' -deoxynuelcotides, which are strong blocks to mammal ian DNA and RNA polymerases, represent a novel class of oxidative DNA lesio n in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of P-32-postlabeled DNA digests have detec ted several bulky oxidative lesions of unknown structure, called I-compound s, in DNA from normal mammalian organs. We investigated whether any of thes e type II I-compounds contained 8,5 ' -cyclo-2 ' -deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of t he sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the techniq ue resulted in detection of two additional I-compounds, pTp-cAp and pGp-eAp . Each I-compound isolated from neonatal rat liver DNA matched authentic P- 32-labeled cA-containing chromatographic standards under nine different chr omatographic conditions. Their levels increased significantly after normal birth. The P-32-postlabeling technique used here is capable of detecting 1- 5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in ti ssues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.