Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants

Abstract

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...

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…E. coli endonuclease III and endonuclease VIII, for example, both recognize the disruption of the helix that results from saturation of pyrimidine residues. While single nth and nei mutants are not noticeably sensitive to H 2 O 2 , the double mutants are rapidly killed (87,88). Endonucleases IV and III complete the repair process by incising the baseless sites, and they also address frank strand breaks that are produced by ribose oxidation.…”

Several pathways of hydrogen peroxide action that damage the E. coli genome

“…The nth nei double mutants are hypersensitive to H 2 O 2 (Jiang et al, 1997). Moreover, endonuclease VIII can recognize 8-oxo-7,8-dihydroxyguanine (8-oxoG) lesions, a kind of lesion also recognized by Fpg protein (Blaisdell et al, 1999).…”

“…Endonuclease IX recognizes urea residues and β-ureidoisobutyric acid in DNA; however, DNA containing thymine glycol or dihydrothymine is not a substrate for this enzyme (Friedberg et al, 1995). Later, the gene for endonuclease VIII (nei) was isolated (Jiang et al, 1997;Saito et al, 1997). Endonuclease VIII is present in extracts of E. coli at 5 to 10% of the level of endonuclease III and is responsible for the repair of 10% of the thymine glycol in E. coli Wallace, 1988).…”

Pathogen DNA as target for host-generated oxidative stress: Role for repair of bacterial DNA damage in Helicobacter pylori colonization

“…nth single mutants exhibit a weak mutator phenotype but are not hypersensitive to DNA-damaging agents able to produce the substrates for this enzyme (17). However, nth nei double mutants show a strong mutator phenotype and are hypersensitive to H 2 O 2 and x-rays (18,19). The product of nei is EndoVIII, a DNA glycosylase that shares a common range of substrates with Nth (19).…”