Mapping Oxidative DNA Damage Using Ligation-Mediated Polymerase Chain Reaction Technology

Abstract

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5′-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 μg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-μg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 μg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 μM H2O2.