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

Abstract

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...

A quantitative assay for assessing the effects of DNA lesions on transcription

“…Owing to the lack of effect of N 2 - dG lesions on DNA replication, this type of lesions have been suggested to be “stealth lesions” 46 . The results from the present study, along with previous in vitro studies 46,47 , showed that these minor-groove N 2 - dG lesions are able to strongly inhibit DNA transcription when present on the DNA template strand. N 2 -ethyl-2′-deoxyguanosine ( N 2 -EtdG) was found, from primer extension assays, to be a strong block to both single-subunit T7 RNAP and multi-subunit RNAPs in vitro 47 .…”

“…The results from the present study, along with previous in vitro studies 46,47 , showed that these minor-groove N 2 - dG lesions are able to strongly inhibit DNA transcription when present on the DNA template strand. N 2 -ethyl-2′-deoxyguanosine ( N 2 -EtdG) was found, from primer extension assays, to be a strong block to both single-subunit T7 RNAP and multi-subunit RNAPs in vitro 47 . In addition, mammalian RNAPII was found to exclusively incorporate the correct nucleotide opposite N 2 -EtdG 47 .…”

“…N 2 -ethyl-2′-deoxyguanosine ( N 2 -EtdG) was found, from primer extension assays, to be a strong block to both single-subunit T7 RNAP and multi-subunit RNAPs in vitro 47 . In addition, mammalian RNAPII was found to exclusively incorporate the correct nucleotide opposite N 2 -EtdG 47 . Likewise, N 2 -furfuryl-dG, a structural mimic of the principal nitrofurazone-induced dG lesions, absolutely blocks transcription by E. coli RNAP when located in the template DNA strand 46 .…”

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

“…Formaldehyde, an environmental concern, reacts with the exocyclic amino group of deoxyguanosine (G), to produce N 2 -methylG [16]. Ethanol is enzymatically oxidized to acetaldehyde, which forms N 2 -ethylG, which has been observed in liver DNA and urine of alcoholic patients [17]. With both of these reactions [13, 14], 2-electron reduction of the intermediate imine is required to balance the stoichiometry.…”

Pathways for repairing and tolerating the spectrum of oxidative DNA lesions

“…RNAPII is generally blocked by more bulky base lesions, e.g. , N 2 -ethyldeoxyguanosine, although the RNA polymerase can insert a C opposite such modifications in some instances [55]. Interference of RNA polymerase progression during transcription has been shown to activate certain cell death responses, and may be particularly relevant to non-dividing cell populations, such as neurons, and thus in diseases of the brain [56].…”