ISOLATION AND CHARACTERIZATION OF ENDONUCLEASE-VIII FROM ESCHERICHIA-COLI

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC(1) purification. The enzyme was found i n strains that contained or lacked endonuclease III and was purified b y radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FP LC. Examination of the properties of endonuclease VIII showed it to ha ve many similarities to endonuclease III. DNA containing thymine glyco l, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP si tes was incised by the enzyme; however, DNA containing reduced AP site s was not. HPLC analysis of the products formed by exhaustive enzymati c digestion of damage-containing DNA showed that endonuclease VIII rel eased thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclea se VIII was not a good substrate for E. coli DNA polymerase I, suggest ing that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after trea ting thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28 000 and 30 000 Da, respectively. SDS-PAGE, in conjunction with activit y gel analysis, gave a molecular mass of about 29 000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to a n active enzyme.