PMID:2453510
| Citation |
Levin, JD, Johnson, AW and Demple, B (1988) Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J. Biol. Chem. 263:8066-71 |
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| Abstract |
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 degrees C by either EDTA or 1,10-phenanthroline, 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 Km of 5-10 nM. The optimal KCl 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 H2O2-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. |
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| Keywords |
DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Endodeoxyribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Free Radicals; Kinetics; Molecular Weight; Oxidation-Reduction; Potassium Chloride/pharmacology; Sodium Chloride/pharmacology |
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