查看文章

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/ H₂O₂ were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H₂O₂ without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate …