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 RNAPII. We also include models of the N(2)-Et-dG within the active site of yeast RNAPII, which are compatible with our observations.
Highlights
Acetaldehyde, the first metabolite of ethanol, reacts with DNA to form adducts, including N2-ethyl-2-deoxyguanosine (N2-Et-dG)
Using primer extension assays in vitro, we found that a single N2-Et-dG lesion is a strong block to both mammalian RNAPII and two other multisubunit RNA polymerases (RNAPs), as well as to T7 RNAP
Using the mammalian RNAPII, we found that CMP is exclusively incorporated opposite the N2-Et-dG lesion
Summary
The sequence of the DNA template strand used in the in vitro ribonucleotide(s) (final concentration, 100 M) and stopped by transcription was: 5Ј-CATGCTGATGAATTCCTTCNCTA- removing samples and mixing with 2ϫ formamide gel loading. RNA oligonucleotide used for running start transcription was raphy. The sequence of the 30-mer Km is the concentration of NTP at which the rate of incorporaRNA marker was 5Ј-UAGGUUCCACCUUACCAGCCUU- tion opposite dG or N2-Et-dG is half-maximal during a fixed. To determine the apparent Km values for RNA Primer Labeling with [␥-32P]ATP by T4 Polynucleotide CTP incorporation opposite dG or N2-Et-dG, standing start. Kinase—RNA primers were labeled with 30 Ci of [␥-32P]ATP experiments were performed with varying concentrations of (PerkinElmer Life Sciences) at 5Ј end by T4 polynucleotide CTP as shown, with incubation for 20 min at 25 °C. The unincorporated nucleotides were removed by NucAway the background values at time 0 [27]. The values given are the means Ϯ S.E., based on two independent determinations
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