Mapping Reactive Oxygen-Induced DNA Damage at Nucleotide Resolution

Abstract

The frequency of reactive oxygen (ROS)-induced DNA base damage along the human p53 and PGK1 genes was determined at nucleotide resolution by cleaving DNA at ROS-modified bases with the Nth and Fpg proteins from Escherichia coli and then using the ligation-mediated PCR (LMPCR) technique to map induced break frequency. Damage was induced either in vivo by exposing cultured human fibroblasts to H2O2 or in vitro by exposing purified genomic DNA to H2O2 /ascorbate in the presence of Cu(II), Fe (III), or Cr(VI) metal ions. With the exception of a few footprints observed in the promoter region of PGK1 at transcription factor binding sites, all four base damage patterns from either in vivo or in vitro treatments were nearly identical in both regions of the genome. Guanines in the triplet d(pCGC) were the most commonly damaged base. Isolated nuclei suffered little ROS-induced DNA base damage in the presence of ascorbate and H2O2; damage was restored by addition of transition metal ions, suggesting that during in vivo exposure of cells to H2O2, metal ions (or metal-like ligands) are freed from extranuclear sites to supply redox-cycling ligands to the nucleus. These data simplify the complexity of H2O2-induced DNA damage and mutagenesis studies by demonstrating the commonality of damage catalyzed by different transition metal ions and by showing that the pattern of H2O2-induced DNA base damage is determined almost entirely by the primary DNA sequence, with chromatin structure having a limited effect.