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(Received for publication, February 10, 1995; and in revised form, May 25, 1995) From the 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/H
Volume 270,
Number 29,
Issue of July 21, pp. 17633-17640, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
O caused DNA base damage by a
sequence-dependent mechanism, with the 5` bases of
d(pG) and d(pC
) 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)-H
O complex, these data suggest that
the local DNA sequence affects formation of
DNA-Cu(I)-HO complexes and/or the efficiency of
base oxidation during resolution of this complex.
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