Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.Free radicals are produced in cells by ionizing radiation, a variety of chemical agents, and normal oxidative metabolism. The spectrum of free radical-induced damage to DNA is broad and includes a wide variety of modifications to the purine and pyrimidine bases, sites of base loss and single-strand breaks (for reviews, see references 11, 39, and 66). The 5,6 double bond of pyrimidines is particularly vulnerable to free radical attack (66), and for DNA thymine, the products include 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol [Tg]), 5-hydroxy-5,6-dihydrothymine, 6-hydroxy-5,6-dihydrothymine, and 5,6-dihydrothymine (produced under anaerobic conditions). 5-Hydroxymethyluracil (24) and a number of ring contraction and fragmentation products such as 5-hydroxy-5-methylhydantoin, methyltartronyl urea, and urea are also formed. A major product of free radical attack on cytosine is 5,6-dihydroxy-5,6-dihydrocytosine (cytosine glycol) (67), which is unstable and dehydrates to form 5-hydroxycytosine (5-OHC) or deaminates to form uracil glycol (Ug), which can also dehydrate to form 5-hydroxyuracil (5-OHU). Contraction and fragmentation products can also be found after free radical attack on DNA cytosine. Of the above-mentioned structures that retain intact ring conformation and base-pairing capabilities, only Tg has been shown to be a potentially lethal lesion. Tg is a potent block to DNA synthesis in vitro (17,28,31,56), using a variety of DNA polymerases, and when present in biologically active single-stranded phage DNA molecules is a lethal lesion with an inactivation efficiency of 1 (1, 29). Tg is also lethal in duplex X DNA, where it takes about 10 to 12 Tg lesions to kill (41). Other intact pyrimidine products su...

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC1 purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released 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 endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating 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 activity gel analysis, gave a molecular mass of about 29,000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, ␤-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (

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