Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: Relating biological consequences to conformational preferences

Thomas M. Schinecker, Rebecca A. Perlow, Suse Broyde, Nicholas E. Geacintov, David A. Scicchitano

Research output: Contribution to journalArticle

Abstract

Environmental polycylic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

Original languageEnglish (US)
Pages (from-to)6004-6015
Number of pages12
JournalNucleic Acids Research
Volume31
Issue number20
DOIs
StatePublished - Oct 15 2003

Fingerprint

DNA Adducts
RNA Polymerase II
Epoxy Compounds
Aromatic Hydrocarbons
DNA-Directed RNA Polymerases
DNA
Nucleotides
Adenine
DNA Repair
Catalytic Domain
benzo(c)phenanthrene

ASJC Scopus subject areas

  • Genetics

Cite this

@article{82c05cbfb4e84338bd8d8cd6964c3528,
title = "Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: Relating biological consequences to conformational preferences",
abstract = "Environmental polycylic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.",
author = "Schinecker, {Thomas M.} and Perlow, {Rebecca A.} and Suse Broyde and Geacintov, {Nicholas E.} and Scicchitano, {David A.}",
year = "2003",
month = "10",
day = "15",
doi = "10.1093/nar/gkg771",
language = "English (US)",
volume = "31",
pages = "6004--6015",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "20",

}

TY - JOUR

T1 - Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides

T2 - Relating biological consequences to conformational preferences

AU - Schinecker, Thomas M.

AU - Perlow, Rebecca A.

AU - Broyde, Suse

AU - Geacintov, Nicholas E.

AU - Scicchitano, David A.

PY - 2003/10/15

Y1 - 2003/10/15

N2 - Environmental polycylic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

AB - Environmental polycylic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

UR - http://www.scopus.com/inward/record.url?scp=0345305415&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0345305415&partnerID=8YFLogxK

U2 - 10.1093/nar/gkg771

DO - 10.1093/nar/gkg771

M3 - Article

VL - 31

SP - 6004

EP - 6015

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 20

ER -