O6-Methylguanine (O6-meG) is a major mutagenic, carcinogenic and cytotoxic DNA adduct produced by various endogenous and exogenous methylating agents. We report the results of transcription past a site-specifically modified O6-meG DNA template by bacteriophage T7 RNA polymerase and human RNA polymerase II. These data show that O6-meG partially blocks T7 RNA polymerase and human RNA polymerase II elongation. In both cases, the sequences of the truncated transcripts indicate that both polymerases stop precisely at the damaged site without nucleotide incorporation opposite the lesion, while extensive misincorporation of uracil is observed in the full-length RNA. For both polymerases, computer models suggest that bypass occurs only when O6-meG adopts an anti conformation around its glycosidic bond, with the methyl group in the proximal orientation; in contrast, blockage requires the methyl group to adopt a distal conformation. Furthermore, the selection of cytosine and uracil partners opposite O6-meG is rationalized with modeled hydrogen-bonding patterns that agree with experimentally observed O6-meG:C and O6-meG:U pairing schemes. Thus, in vitro, O6-meG contributes substantially to transcriptional mutagenesis. In addition, the partial blockage of RNA polymerase II suggests that transcription-coupled DNA repair could play an auxiliary role in the clearance of this lesion.
Damage in transcribed DNA presents a challenge to the cell because it can partially or completely block the progression of an RNA polymerase, interfering with transcription and compromising gene expression. While blockage of RNA polymerase progression is thought to trigger the recruitment of transcription-coupled DNA repair (TCR), bypass of the lesion can also occur, either error-prone or error-free. Error-prone transcription is often referred to as transcriptional mutagenesis (TM). Elucidating why some lesions pose blocks to transcription elongation while others do not remains a challenging problem. As part of an effort to understand this, we studied transcription past a 5-guanidino-4-nitroimidazole (NI) lesion, using two structurally different RNA polymerases, human RNA polymerase II (hRNAPII) and bacteriophage T7 RNA polymerase (T7RNAP). The NI damage results from the oxidation of guanine in DNA by peroxynitrite, a well known, biologically important oxidant. It is of structural interest because it is a ring-opened and conformationally flexible guanine lesion. Our results show that NI acts as a partial block to T7RNAP while posing a major block to hRNAPII, which has a more constrained active site than T7RNAP. Lesion bypass by T7RNAP induces base misincorporations and deletions opposite the lesion (C>A>-1 deletion >G >>> U), but hRNAPII exhibits error-free transcription although lesion bypass is a rare event. We employed molecular modeling methods to explain the observed blockage or bypass accompanied by nucleotide incorporation opposite the lesion. The results of the modeling studies indicate that NI's multiple hydrogen-bonding capabilities and torsional flexibility are important determinants of its effect on transcription in both enzymes. These influence the kinetics of lesion bypass and may well play a role in TM and TCR in cells.
SummaryThe DNA lesion 1,N 2 -ethenoguanine is formed endogenously as a byproduct of lipid peroxidation or by reaction with epoxides that result from the metabolism of the industrial pollutant vinyl chloride, a known human carcinogen. DNA replication past 1,N 2 -ethenoguanine and site specific mutagenesis studies in mammalian cells have established the highly mutagenic and genotoxic properties of the damaged base. However, there is as yet no information on the processing of this lesion during transcription. Here, we report the results of transcription past a site specifically modified 1,N 2 -ethenoguanine DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen bonding edge of guanine. Our results show that 1,N 2 -ethenoguanine acts as a partial block to the bacteriophage T7 RNA polymerase, which allows nucleotide incorporation in the growing RNA with the selectivity A > G > (C = −1 deletion) >> U. In contrast, 1,N 2 -ethenoguanine poses an absolute block to human RNA polymerase II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNA polymerase allows lesion bypass when the 1,N 2 -ethenoguanine adopts the syn conformation. This orientation places the exocylic ring in a collision free empty pocket of the polymerase, and the observed base incorporation preferences are in agreement with hydrogen bonding possibilities between the incoming nucleotides and the Hoogsteen edge of the lesion. On the other hand, in the more crowded active site of the human RNA polymerase II, the modeling studies show that both syn and anti conformations of the 1,N 2 -ethenoguanine are sterically impermissible. Polymerase stalling is currently believed to trigger the transcription coupled nucleotide excision repair machinery. Thus, our data suggest that this repair pathway likely is engaged in the clearance of the 1,N 2 -ethenoguanine from actively transcribed DNA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.