Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA (original) (raw)

1988, Journal of Biological Chemistry

Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis. Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene. Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E. coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat. Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 "C by either EDTA or 1,lOphenanthroline, suggesting the presence of an essential metal tightly bound to the protein. Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent K, of 5-10 nM. The optimal KC1 or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM. The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities. Endonuclease IV also activated H,Oz-damaged DNA for repair synthesis by DNA polymerase I. Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity. Partially reduced oxygen species are toxic and mutagenic to cells (Halliwell, 1987). These species, notably superoxide radical anion (O;), hydrogen peroxide, and hydroxyl radical (HO), are formed by ionizing radiation (Hutchinson, 1985), antibiotics such as paraquat and streptonigrin (Hassett et al., 1987), and by normal cellular metabolism (Fridovich, 1978). Living cells have a front-line enzymatic defense system against damage by oxygen radicals in the form of superoxide dismutase and catalase-hydroperoxidase (Fridovich, 1978). When these scavenging enzymes are missing (Farr et al., 1986) or noninducible (Storz et al., 1987), the spontaneous mutation frequency in Escherichia coli and Salmonella typhimurium is elevated during aerobic growth, while overproduction of per-* This work was supported by Grants CA37831 and ES03926 from the National Institutes of Health (to B. D.