DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner

Rebecca A. Perlow, Alexander Kolbanovskii, Brian E. Hingerty, Nicholas E. Geacintov, Suse Broyde, David A. Scicchitano

Research output: Contribution to journalArticle

Abstract

Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]-pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N2-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N2-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)-and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.

Original languageEnglish (US)
Pages (from-to)29-47
Number of pages19
JournalJournal of Molecular Biology
Volume321
Issue number1
DOIs
StatePublished - 2002

Fingerprint

DNA Adducts
RNA Polymerase II
Benzo(a)pyrene
Carcinogens
DNA
Epoxy Compounds
DNA Repair
Polycyclic Aromatic Hydrocarbons
Genome
Environmental Carcinogens
Aptitude
Mixed Function Oxygenases
Catalytic Domain
Neoplasms
RNA
Mutation
Enzymes
Genes

Keywords

  • Benzo[a]pyrene
  • DNA adduct stereochemistry
  • RNA polymerase II
  • Transcription
  • Transcription junction structure

ASJC Scopus subject areas

  • Virology

Cite this

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title = "DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner",
abstract = "Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]-pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N2-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N2-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)-and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.",
keywords = "Benzo[a]pyrene, DNA adduct stereochemistry, RNA polymerase II, Transcription, Transcription junction structure",
author = "Perlow, {Rebecca A.} and Alexander Kolbanovskii and Hingerty, {Brian E.} and Geacintov, {Nicholas E.} and Suse Broyde and Scicchitano, {David A.}",
year = "2002",
doi = "10.1016/S0022-2836(02)00593-4",
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TY - JOUR

T1 - DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner

AU - Perlow, Rebecca A.

AU - Kolbanovskii, Alexander

AU - Hingerty, Brian E.

AU - Geacintov, Nicholas E.

AU - Broyde, Suse

AU - Scicchitano, David A.

PY - 2002

Y1 - 2002

N2 - Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]-pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N2-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N2-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)-and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.

AB - Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]-pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N2-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N2-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)-and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.

KW - Benzo[a]pyrene

KW - DNA adduct stereochemistry

KW - RNA polymerase II

KW - Transcription

KW - Transcription junction structure

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