Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA

Abstract

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas, bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO·, and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. There results inddicate a metal-catalyzed interaction of O 2 − with H 2O 2, which produces HO· which, in turn, causes DNA strand scission and oxidation of thymine residues III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O 2 − prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O 2 − production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O 2 − was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O 2 − plus H 2O 2 both in vitro and in vivo. to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease