A 32P-Postlabeling Assay for the Oxidative DNA Lesion 8,5′-Cyclo-2′-deoxyadenosine in Mammalian Tissues
8,5′-Cyclopurine-2′-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies usi...
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Veröffentlicht in: | The Journal of biological chemistry 2001-09, Vol.276 (38), p.36051-36057 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | 8,5′-Cyclopurine-2′-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion 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 32P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5′-cyclo-2′-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic32P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The 32P-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 tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M105472200 |