DNA damage by peroxynitrite characterized with DNA repair enzymes.
The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relativ…
[33] Use of repair endonucleases to assess DNA damage by peroxynitrite
Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by peroxynitrite. Repair endonucleases allow a convenient quantification of various types of oxidative modifications induced by peroxynitrite, both in cultured cells and in cell-free DNA. The high sensitivity of the assays allows highly ectotoxic exposure conditions to be avoided—as well as the generation of secondary DNA modifications—that often become a problem at high levels of damage because primary DNA oxidation products can be orders of magnitude more reactive than the original bases, as demonstrated for the reaction of 8-hydroxyguanine with singlet oxygen. The ratio of the various types of m…