Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli

Upton, Dana C.; Blans, Patrick; Perrino, Fred W.; Fishbein, James C.; Akman, Steven A.

doi:10.1016/j.mrfmmm.2005.12.014

Cited by 17 publications

(15 citation statements)

References 30 publications

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“…In addition, cells have specialized DNA polymerases that can bypass N 2 -Et-dG in an error-free manner (16,19,20). In human cells in vivo, the major genotoxic effects of the lesion appear to be the result of replication blockage, with some capacity for generating mutations including single base deletions and transversions (21).…”

Section: Acetaldehyde (Acd)mentioning

confidence: 99%

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Cheng

Gnatt

et al. 2008

Journal of Biological Chemistry

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Acetaldehyde, the first metabolite of ethanol, reacts with DNA to form adducts, including N 2 -ethyl-2-deoxyguanosine (N 2 -Et-dG). Although the effects of N 2 -Et-dG on DNA polymerases have been well studied, nothing is known about possible effects of this lesion on transcription by RNA polymerases (RNAPs). Using primer extension assays in vitro, we found that a single N 2 -Et-dG lesion is a strong block to both mammalian RNAPII and two other multisubunit RNAPs, (yeast RNAPII and Escherichia coli RNAP), as well as to T7 RNAP. However, the mechanism of transcription blockage appears to differ between the multisubunit RNAPs and T7 RNAP. Specifically, all three of the multisubunit RNAPs can incorporate a single rNTP residue opposite the lesion, whereas T7 RNAP is essentially unable to do so. Using the mammalian RNAPII, we found that CMP is exclusively incorporated opposite the N 2 -Et-dG lesion. In addition, we also show that the accessory transcription factor TFIIS does not act as a lesion bypass factor, as it does for other nonbulky DNA lesions; instead, it stimulates the polymerase to remove the CMP incorporated opposite the lesion by mammalian RNA-PII. We also include models of the N 2 -Et-dG within the active site of yeast RNAPII, which are compatible with our observations. Acetaldehyde (ACD)2 is a genotoxin, known animal carcinogen, and suspected human carcinogen (1, 2). Although small amounts of ACD are produced endogenously during threonine catabolism (3), the most significant source of human exposure to ACD is via the metabolism of ethanol. In the human body, ethanol is first converted to ACD via the enzyme alcohol dehydrogenase, and ACD is further converted to acetate via aldehyde dehydrogenase (ALDH), primarily by the hepatic enzyme ALDH2. Approximately 50% of East Asian individuals are deficient in ALDH2 activity because of an amino acid substitution resulting in an inactive enzyme (4). ALDH2-deficient individuals are at a substantially elevated risk of esophageal cancer when they drink heavily, and other mechanistic evidence indicates that ACD is responsible for the increased cancer risk (2).Several studies have shown that ACD can react with DNA to form adducts (5-12). One of the first identified and most well studied ACD-derived lesions is N 2 -ethyl-2Ј-deoxyguanosine (N 2 -Et-dG) (Fig. 1). N 2 -Et-dG is the stable form of N 2 -Eti-dG, the immediate product of the ACD reaction with dG. In the presence of basic compounds such as histones and polyamines, ACD can also give rise to other DNA adducts (13,14). Elevated levels of these ACD-related DNA adducts, including N 2 -Et-dG, have been observed in white blood cell DNA in humans following alcohol consumption, with significantly higher levels observed in ALDH2-deficient individuals (15). Thus, the biological effects of these DNA lesions are of potential clinical relevance.In view of the relationship between alcohol and cancer, the effect of N 2 -Et-dG on DNA replication and mutagenesis have been well studied. N 2 -Et-dG is a strong block to DNA polymera...

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Section: Acetaldehyde (Acd)mentioning

confidence: 99%

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Cheng

Gnatt

et al. 2008

Journal of Biological Chemistry

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“…These data suggest that N 2 -ethyldGuo might induce G:C f C:G transversions in vivo (12). However, the mutation spectrum of a single chemically synthesized N 2 -ethyldGuo adduct serving as a template in E. coli in vivo consisted of G:C f T:A transversions and single base deletions, at either the adduct site or 3-5 nucleotides downstream of the adduct (16).…”

Section: Introductionmentioning

confidence: 96%

Replication of N²-Ethyldeoxyguanosine DNA Adducts in the Human Embryonic Kidney Cell Line 293

Upton

Blans

Perrino

et al. 2006

Chem. Res. Toxicol.

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N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX. N(2)-EthyldGuo and O(6)-ethyldGuo were both minimally mutagenic in double-stranded pLSX replicated in human 293 cells; however, the placement of deoxyuridines on the complementary strand at 5'- and 3'-positions flanking the adduct resulted in 5- and 22-fold enhancements of the N(2)-ethyldGuo- and O(6)-ethyldGuo-induced mutant fractions, respectively. The fold increase in the N(2)-ethyldGuo-induced mutant fraction in deoxyuridine-containing plasmids was similar after replication in 293T cells, a mismatch repair deficient variant of 293 cells, indicating that postreplication mismatch repair has little role in modulating N(2)-ethyldGuo-mediated mutagenesis. The mutation spectrum generated by N(2)-ethyldGuo consisted primarily of single base deletions and adduct site-targeted transversions, in contrast to the exclusive production of adduct site-targeted transitions by O(6)-ethyldGuo. The yield of progeny plasmids after replication in 293 cells was reduced by the presence of N(2)-ethyldGuo in parental plasmids with or without deoxyuridine to 39 or 19%, respectively. Taken together, these data indicate that N(2)-ethyldGuo in DNA exerts its principal biological activity by blocking translesion DNA synthesis in human cells, resulting in either failure of replication or frameshift deletion mutations.

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“…N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA pol I (9). Mutations caused by N 2 -ethyl-Gua range from single base deletions to transversions (10).…”

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confidence: 99%

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

Pence

Blans

Zink

et al. 2009

Journal of Biological Chemistry

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Nucleotide incorporation and extension opposite N 2 -ethylGua by DNA polymerase was measured and structures of the DNA polymerase -N 2 -ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase opposite N 2 -ethyl-Gua was determined by steady state kinetic analysis with Mg 2؉ or Mn 2؉ as the activating metal. DNA polymerase incorporates dCMP opposite N 2 -ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg 2؉ and with greater efficiencies in the presence of Mn 2؉ . However, the fidelity of nucleotide incorporation by DNA polymerase opposite N 2 -ethyl-Gua and Gua using Mn 2؉ is lower relative to that using Mg 2؉ indicating a metal-dependent effect. DNA polymerase extends from the N 2 -ethyl-Gua:Cyt 3 terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase catalyzed reaction is well suited for N 2 -ethyl-Gua bypass. The structure of DNA polymerase with N 2 -ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase complexed with N 2 -ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.2 is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2Ј-deoxyguanosine-3Ј-monophosphate (1). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2). N 2 -Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2, 3). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4). The formation of N 2 -ethylGua during the reduction of acetaldehyde could cause ethanolrelated cancers (5).The ethyl moiety of N 2 -ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6, 7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8). The blocking of replicative DNA pols by N 2 -ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8). N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA po...

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Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli

Cited by 17 publications

References 30 publications

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Replication of N²-Ethyldeoxyguanosine DNA Adducts in the Human Embryonic Kidney Cell Line 293

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

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Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli

Cited by 17 publications

References 30 publications

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Differential Blocking Effects of the Acetaldehyde-derived DNA Lesion N2-Ethyl-2′-deoxyguanosine on Transcription by Multisubunit and Single Subunit RNA Polymerases

Replication of N2-Ethyldeoxyguanosine DNA Adducts in the Human Embryonic Kidney Cell Line 293

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

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Replication of N²-Ethyldeoxyguanosine DNA Adducts in the Human Embryonic Kidney Cell Line 293