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Metal Ion-dependent Hydrogen Peroxide-induced DNA Damage Is More Sequence Specific than Metal Specific¹

Department of Medical Oncology and Therapeutics Research [H. R., S. A. A.], and Division of Biology, Beckman Research Institute [G. P. H.], City of Hope National Medical Center, Duarte, California 91010; and Department of Biostatistics, Wake Forest Comprehensive Cancer Center, Winston-Salem, North Carolina 27157 [R. D., J. K.]

The frequency of oxidative base damage along the human p53 and PGK1 genes was determined at nucleotide resolution by cleaving DNA at oxidized bases with endonuclease III and formamidopyrimidine DNA glycosylase and then using the ligation-mediated PCR technique to map induced break frequency. Damage was induced either in vivo by exposing cultured human male fibroblasts to H₂O₂ or in vitro by exposing purified genomic DNA to H₂O₂ plus ascorbate in the presence of Cu(II), Fe(III), or Cr(VI) metal ions. All four base damage patterns from either in vivo or in vitro treatments were nearly identical in both regions of the genome. The frequency of base damage varied along the DNA, with guanine being the most commonly damaged base. In the Fe(III)-mediated in vitro reactions, single-stranded breaks were almost completely suppressed by addition of sucrose, which facilitated mapping of base damage. The in vitro base damage pattern generated by Cr(VI), ascorbate, and H₂O₂ was similar to that of the other metal ions, with the exception of several unique positions; these were heavily damaged only in the presence of Cr(VI). Isolated nuclei suffered little oxidative base damage in the presence of ascorbate and H₂O₂, and we conclude that during H₂O₂ in vivo treatment of cells, metal ions (or metal-like ligands) are freed from the cytoplasm to migrate into the nucleus and supply the redox cycling ligands necessary for oxidative base damage. These data simplify the complexity of H₂O₂-induced oxidative damage and mutagenesis studies by demonstrating the commonality of damage catalyzed by different transition metal ions and by showing that the pattern of H₂O₂-mediated oxidative base damage is determined almost entirely by the primary DNA sequence, with chromatin structure having a limited effect. Our data suggest a model for base damage in which DNA-metal ion binding domains can equally accommodate a variety of different metal ions and thus are a key factor in determining the local probability of DNA damage.

¹ This work was supported by United States Public Health Service Grant CA-53115 from the National Cancer Institute (to S. A. A.).

² To whom requests for reprints should be addressed, at Department of Cancer Biology, Comprehensive Cancer Center of Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157. Tel: (910) 716-0230; Fax: (910) 716-0255; E-mail: sakman@bgsm.edu.

Received 8/ 5/96. Accepted 4/21/97.

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