Visualizing sequence-governed nucleotide selectivities and mutagenic consequences through a replicative cycle: Processing of a bulky carcinogen N2-dG lesion in a Y-family DNA polymerase

Pingna Xu, Lida Oum, Yuan Cho Lee, Nicholas E. Geacintov, Suse Broyde

Research output: Contribution to journalArticle

Abstract

Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S-(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5′-C[BP]G*G-3′ sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5′-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5′-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

Original languageEnglish (US)
Pages (from-to)4677-4690
Number of pages14
JournalBiochemistry
Volume48
Issue number22
DOIs
StatePublished - Jun 9 2009

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DNA-Directed DNA Polymerase
Carcinogens
Nucleotides
Catalytic Domain
Processing
Molecular modeling
Benzo(a)pyrene
Molecular Dynamics Simulation
Molecular dynamics
Kinetics
Computer simulation
deoxyguanosine triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{22045023ca0646fdb53d99dd9cb4ab7b,
title = "Visualizing sequence-governed nucleotide selectivities and mutagenic consequences through a replicative cycle: Processing of a bulky carcinogen N2-dG lesion in a Y-family DNA polymerase",
abstract = "Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S-(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5′-C[BP]G*G-3′ sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5′-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5′-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.",
author = "Pingna Xu and Lida Oum and Lee, {Yuan Cho} and Geacintov, {Nicholas E.} and Suse Broyde",
year = "2009",
month = "6",
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journal = "Biochemistry",
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publisher = "American Chemical Society",
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T1 - Visualizing sequence-governed nucleotide selectivities and mutagenic consequences through a replicative cycle

T2 - Processing of a bulky carcinogen N2-dG lesion in a Y-family DNA polymerase

AU - Xu, Pingna

AU - Oum, Lida

AU - Lee, Yuan Cho

AU - Geacintov, Nicholas E.

AU - Broyde, Suse

PY - 2009/6/9

Y1 - 2009/6/9

N2 - Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S-(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5′-C[BP]G*G-3′ sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5′-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5′-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

AB - Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S-(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5′-C[BP]G*G-3′ sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5′-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5′-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

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