In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide adducts

Parvathi Chary, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3′ to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in ∼16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3′→5′ exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.

Original languageEnglish (US)
Pages (from-to)1398-1405
Number of pages8
JournalNucleic Acids Research
Volume23
Issue number8
StatePublished - Apr 25 1995
Externally publishedYes

Fingerprint

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Pyrene
Bearings (structural)
DNA-Directed DNA Polymerase
Replication
Template
DNA
Carcinogens
Bacteriophages
Elongation
Environmental Carcinogens
DNA Polymerase I
Bacteriophage T4
Exonucleases
Substrates
Synthesis
DNA Adducts
Single-Stranded DNA
Adenine
Configuration

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

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title = "In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide adducts",
abstract = "DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3′ to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in ∼16{\%} full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3′→5′ exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.",
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AU - Lloyd, Robert (Stephen)

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