Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

133

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-Å crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5 template guanine. The position of the displaced 5 template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.oxidative DNA damage ͉ structure ͉ DNA replication T hymine glycol (5,6-dihydro-5,6-dihydroxythymine; Tg), the most common oxidation product of thymine, is produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as chemical oxidants or ionizing radiation (Fig. 1). It is estimated that 400 Tgs are formed per cell per day (1, 2), and the presence of Tg in DNA has been used as a marker for oxidative stress (1, 3). Moreover, Tg is one of the predominant types of base modifications produced by ionizing radiation (4, 5), including that used in cancer therapy.Of the oxidatively modified DNA bases retaining an intact ring, Tg is thought to induce the most distortion in the regular structure of DNA. Even though there are DNA repair enzymes specialized in excising Tg from the genome, such as human , statistically a few of the damaged bases will evade repair, which means that DNA polymerases will encounter these lesions during replication. Although Tg is a poor mutagenic lesion because it generally pairs with A (9), it has been shown to be a very effective block to DNA replication (10-13). When Tg is encountered as a templating base by replicative or repair polymerases, termination of primer extension occurs immediately past the lesion site with A inserted opposite the Tg. Even in the presence of proofreading, extension proceeds no further. This finding contrasts with the situation observed when the lesion is an abasic site, where, in the presence of proofreading, termination sites are observed one base before the lesion (14). Therefore, it is extension past the Tg⅐adenine pair, rather than insertion across Tg, that constitutes the block to the replicative polymerases. Because Tg is a strong block to repair and replicative DNA polymerases in vitro (10-13), not surprisingly, it is a lethal lesion in vivo (9,(15)(16)(17)(18).Unlike normal DNA bases, Tg is nonplanar because of the loss of aromatic character that accompanies the addition of hydroxyl groups at the 5 and 6 positions of the ring (19). Computational simulations (20,21) predict that the axial orientation of the methyl group with respect to the pyrimidine r...

supporting

confidence: 67%

mentioning

confidence: 99%

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A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

Aller

Rould

Hogg³

et al. 2007

133

“…Single-molecule experiments were performed using λ DNA (New England BioLabs). λ DNA containing Tg or oxidized purine was generated by exposure to osmium tetroxide (OsO 4 ) plus heat and MB plus visible light, respectively, as previously described (24)(25)(26)(30)(31)(32). Briefly, 100 μg/mL λ DNA was treated with 0.2 and 1 μg/mL MB and then illuminated for 60 min with one 100-W lamp (Reveal; 100 W; 1,352 Lumens; A-19 Shape; General Electric) that was placed at a distance of 20 cm from the surface of a thinly coated 10-cm plate containing the DNA solution placed on ice.…”

Section: Methodsmentioning

confidence: 99%

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

Nelson

Dunn

Kathe

et al. 2014

113

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.DNA repair | search mechanisms O xidative DNA damage is produced endogenously during normal cellular metabolism or exogenously by chemical agents and ionizing radiation (1, 2). Oxidatively induced DNA damage resulting from attack by reactive oxygen species accounts for approximately one-half of all DNA base damages (3). Some oxidative base damages, such as thymine glycol, are blocks to DNA polymerases and thus potentially lethal; however, the majority of oxidative base lesions mispair with noncognate bases and are potentially mutagenic (4). Therefore, damaged bases must be repaired to maintain the cell's genomic integrity. With substantial in vivo steady-state levels of oxidative damages, alkylation damages, and apurinic/apyrimidinic (AP) sites among the nearly six billion normal bases, how DNA repair enzymes locate these damages in the sea of undamaged bases has been the subject of much speculation.The DNA repair mechanism responsible for the removal of the majority of endogenous DNA damages is the base excision repair (BER) pathway (4-6). The critical first step in BER is carried out by a DNA glycosylase that, fueled only by thermal energy, locates a damaged base and cleaves the N-glycosyl bond, thus removing the base lesion from the sugar phosphate backbone. Glycosylases are small monomeric proteins that are found in all living organisms and can be separated into different families based on substrate specificity and structural motifs. In Escherichia coli, there are three glycosylases, Nth, Fpg, and Nei, that directly remove oxidized DNA bases, and all three have an associated lyase activity that cleaves the DNA backbone. These glycosylases are members of two structural families, the helixhairpin-helix (HhH) or Nth superfamily and the helix-two turnshelix (H2TH) or Fpg/Nei family (7-9) (Fig. 1A). Interestingly, the HhH superfamily member, endonuclease III (Nth), and Fpg/ Nei family member, endonuclease VIII (Nei), primarily catalyze the removal of oxidized pyrimidines, such as 5,6-dihydroxy-5, 6-dihydrothymine (Tg), whereas formamidopyrimidine DNA glycosylase (Fpg) primarily removes oxidized purines...

“…Other common pyrimidine derivatives are 5,6-dihydrothymine and urea (13). In Escherichia coli, endonuclease III (EndoIII) is the enzyme responsible of excising these lethal or mutagenic pyrimidine lesions (16,17). In addition to its DNA glycosylase activity, all of the EndoIII enzymes described possess an abasic (AP) lyase activity that cleaves the DNA backbone 3Ј of the AP site.…”

mentioning

confidence: 99%

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

O’Rourke

Chevalier

Pinto

et al. 2003

122

110

Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.