Translesion synthesis by human DNA polymerase κ on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)

Naomi Suzuki, Eiji Ohashi, Alexander Kolbanovskiy, Nicholas Geacintov, Arthur P. Grollman, Haruo Ohmori, Shinya Shibutani

Research output: Contribution to journalArticle

Abstract

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase κ (pol κ), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of κ (pol κδC), translesion synthesis past dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,-10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N2-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol κ δC. Primer extension was retarded one base prior to the dG-N2-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N2-BPDE lesions. (+)-trans-dG-N2-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N2-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N2-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N2-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (Fins x Fext) of dC·dG-N2-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol κ may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N2-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N2-BPDE adducts in human cells.

Original languageEnglish (US)
Pages (from-to)6100-6106
Number of pages7
JournalBiochemistry
Volume41
Issue number19
DOIs
StatePublished - May 14 2002

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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Stereoisomerism
DNA-Directed DNA Polymerase
DNA
DNA Adducts
9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene
benzo(a)pyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine
Oligodeoxyribonucleotides
DNA Primers
Mutation Rate
Isomers
Escherichia coli
Cells

ASJC Scopus subject areas

  • Biochemistry

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Translesion synthesis by human DNA polymerase κ on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). / Suzuki, Naomi; Ohashi, Eiji; Kolbanovskiy, Alexander; Geacintov, Nicholas; Grollman, Arthur P.; Ohmori, Haruo; Shibutani, Shinya.

In: Biochemistry, Vol. 41, No. 19, 14.05.2002, p. 6100-6106.

Research output: Contribution to journalArticle

Suzuki, Naomi ; Ohashi, Eiji ; Kolbanovskiy, Alexander ; Geacintov, Nicholas ; Grollman, Arthur P. ; Ohmori, Haruo ; Shibutani, Shinya. / Translesion synthesis by human DNA polymerase κ on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). In: Biochemistry. 2002 ; Vol. 41, No. 19. pp. 6100-6106.
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abstract = "Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase κ (pol κ), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of κ (pol κδC), translesion synthesis past dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,-10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N2-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol κ δC. Primer extension was retarded one base prior to the dG-N2-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N2-BPDE lesions. (+)-trans-dG-N2-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7{\%} of the starting primers) and deletions (1.1{\%}). Although (+)-cis-dG-N2-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N2-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N2-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (Fins x Fext) of dC·dG-N2-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol κ may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N2-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N2-BPDE adducts in human cells.",
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AU - Suzuki, Naomi

AU - Ohashi, Eiji

AU - Kolbanovskiy, Alexander

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N2 - Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase κ (pol κ), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of κ (pol κδC), translesion synthesis past dG-(+)- or dG-(-)-anti-N2-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,-10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N2-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol κ δC. Primer extension was retarded one base prior to the dG-N2-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N2-BPDE lesions. (+)-trans-dG-N2-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N2-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N2-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N2-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (Fins x Fext) of dC·dG-N2-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol κ may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N2-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N2-BPDE adducts in human cells.

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