Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DN A can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichi a coli, Fpg glycosylase, and endonuclease iii. The frequency of the resulti ng glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide res olution along a sequencing gel autoradiogram by a genomic sequencing techni que, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat c ells, the frequency of endogenous oxidized bases in mitochondrial DNA is su fficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 mug of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-mug preparations of total cellular DNA. Preparative gel electrophor esis of the PGK1 and p53 genes from 300 mug of restriction endonuclease-dig ested genomic DNA showed a 25-fold enrichment for the genes and, after endo nuclease digestion followed by LMPCR, gave sufficient signal to map the fre quency of oxidized bases from human cells treated with 50 muM H2O2. (C) 200 0 Academic Press.