Transcription Processing at 1,N2-Ethenoguanine by Human RNA Polymerase II and Bacteriophage T7 RNA Polymerase

Alexandra Dimitri, Angela K. Goodenough, F. Peter Guengerich, Suse Broyde, David A. Scicchitano

Research output: Contribution to journalArticle

Abstract

The DNA lesion 1,N2-ethenoguanine (1,N2-εG) is formed endogenously as a by-product 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,N2-εG and site-specific mutagenesis studies on 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,N2-εG DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N2-εG acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A > G > (C = - 1 deletion) ≫ U. In contrast, 1,N2-εG poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N2-εG adopts the syn-conformation. This orientation places the exocyclic 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 RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N2-εG 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 is likely engaged in the clearance of the 1,N2-εG from actively transcribed DNA.

Original languageEnglish (US)
Pages (from-to)353-366
Number of pages14
JournalJournal of Molecular Biology
Volume375
Issue number2
DOIs
StatePublished - Jan 11 2008

Fingerprint

RNA Phages
RNA Polymerase II
Nucleotides
Hydrogen Bonding
DNA
Catalytic Domain
Vinyl Chloride
1,(N2)-ethenoguanine
bacteriophage T7 RNA polymerase
Epoxy Compounds
Guanine
Site-Directed Mutagenesis
DNA Replication
Automatic Data Processing
DNA Repair
Carcinogens
Lipid Peroxidation
RNA

Keywords

  • 1,N-ethenoguanine lesion
  • DNA damage
  • molecular modeling
  • RNA sequencing
  • transcription

ASJC Scopus subject areas

  • Virology

Cite this

Transcription Processing at 1,N2-Ethenoguanine by Human RNA Polymerase II and Bacteriophage T7 RNA Polymerase. / Dimitri, Alexandra; Goodenough, Angela K.; Guengerich, F. Peter; Broyde, Suse; Scicchitano, David A.

In: Journal of Molecular Biology, Vol. 375, No. 2, 11.01.2008, p. 353-366.

Research output: Contribution to journalArticle

@article{130b7f62e11e4f039eb247bbd56c98a6,
title = "Transcription Processing at 1,N2-Ethenoguanine by Human RNA Polymerase II and Bacteriophage T7 RNA Polymerase",
abstract = "The DNA lesion 1,N2-ethenoguanine (1,N2-εG) is formed endogenously as a by-product 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,N2-εG and site-specific mutagenesis studies on 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,N2-εG DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N2-εG acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A > G > (C = - 1 deletion) ≫ U. In contrast, 1,N2-εG poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N2-εG adopts the syn-conformation. This orientation places the exocyclic 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 RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N2-εG 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 is likely engaged in the clearance of the 1,N2-εG from actively transcribed DNA.",
keywords = "1,N-ethenoguanine lesion, DNA damage, molecular modeling, RNA sequencing, transcription",
author = "Alexandra Dimitri and Goodenough, {Angela K.} and Guengerich, {F. Peter} and Suse Broyde and Scicchitano, {David A.}",
year = "2008",
month = "1",
day = "11",
doi = "10.1016/j.jmb.2007.10.057",
language = "English (US)",
volume = "375",
pages = "353--366",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Transcription Processing at 1,N2-Ethenoguanine by Human RNA Polymerase II and Bacteriophage T7 RNA Polymerase

AU - Dimitri, Alexandra

AU - Goodenough, Angela K.

AU - Guengerich, F. Peter

AU - Broyde, Suse

AU - Scicchitano, David A.

PY - 2008/1/11

Y1 - 2008/1/11

N2 - The DNA lesion 1,N2-ethenoguanine (1,N2-εG) is formed endogenously as a by-product 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,N2-εG and site-specific mutagenesis studies on 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,N2-εG DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N2-εG acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A > G > (C = - 1 deletion) ≫ U. In contrast, 1,N2-εG poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N2-εG adopts the syn-conformation. This orientation places the exocyclic 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 RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N2-εG 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 is likely engaged in the clearance of the 1,N2-εG from actively transcribed DNA.

AB - The DNA lesion 1,N2-ethenoguanine (1,N2-εG) is formed endogenously as a by-product 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,N2-εG and site-specific mutagenesis studies on 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,N2-εG DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N2-εG acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A > G > (C = - 1 deletion) ≫ U. In contrast, 1,N2-εG poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N2-εG adopts the syn-conformation. This orientation places the exocyclic 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 RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N2-εG 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 is likely engaged in the clearance of the 1,N2-εG from actively transcribed DNA.

KW - 1,N-ethenoguanine lesion

KW - DNA damage

KW - molecular modeling

KW - RNA sequencing

KW - transcription

UR - http://www.scopus.com/inward/record.url?scp=36549024502&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36549024502&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2007.10.057

DO - 10.1016/j.jmb.2007.10.057

M3 - Article

C2 - 18022639

AN - SCOPUS:36549024502

VL - 375

SP - 353

EP - 366

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -