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Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.

Publication ,  Journal Article
Rodriguez, H; Drouin, R; Holmquist, GP; O'Connor, TR; Boiteux, S; Laval, J; Doroshow, JH; Akman, SA
Published in: J Biol Chem
July 21, 1995

The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

July 21, 1995

Volume

270

Issue

29

Start / End Page

17633 / 17640

Location

United States

Related Subject Headings

  • Polymerase Chain Reaction
  • Phosphoglycerate Kinase
  • Nucleotides
  • Molecular Sequence Data
  • Male
  • Hydrogen Peroxide
  • Humans
  • DNA Damage
  • Copper
  • Biochemistry & Molecular Biology
 

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Rodriguez, H., Drouin, R., Holmquist, G. P., O’Connor, T. R., Boiteux, S., Laval, J., … Akman, S. A. (1995). Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J Biol Chem, 270(29), 17633–17640. https://doi.org/10.1074/jbc.270.29.17633
Rodriguez, H., R. Drouin, G. P. Holmquist, T. R. O’Connor, S. Boiteux, J. Laval, J. H. Doroshow, and S. A. Akman. “Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.J Biol Chem 270, no. 29 (July 21, 1995): 17633–40. https://doi.org/10.1074/jbc.270.29.17633.
Rodriguez H, Drouin R, Holmquist GP, O’Connor TR, Boiteux S, Laval J, et al. Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J Biol Chem. 1995 Jul 21;270(29):17633–40.
Rodriguez, H., et al. “Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.J Biol Chem, vol. 270, no. 29, July 1995, pp. 17633–40. Pubmed, doi:10.1074/jbc.270.29.17633.
Rodriguez H, Drouin R, Holmquist GP, O’Connor TR, Boiteux S, Laval J, Doroshow JH, Akman SA. Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J Biol Chem. 1995 Jul 21;270(29):17633–17640.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

July 21, 1995

Volume

270

Issue

29

Start / End Page

17633 / 17640

Location

United States

Related Subject Headings

  • Polymerase Chain Reaction
  • Phosphoglycerate Kinase
  • Nucleotides
  • Molecular Sequence Data
  • Male
  • Hydrogen Peroxide
  • Humans
  • DNA Damage
  • Copper
  • Biochemistry & Molecular Biology