Toward understanding the mutagenicity of an environmental carcinogen: Structural insights into nucleotide incorporation preferences

Rebecca A. Perlow, Suse Broyde

Research output: Contribution to journalArticle

Abstract

Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N2-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be mis-replicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N2-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A > T ≥ G > C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N2-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N2-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N2-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N2-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N2-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N2-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N2-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N2-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N2-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.

Original languageEnglish (US)
Pages (from-to)291-309
Number of pages19
JournalJournal of Molecular Biology
Volume322
Issue number2
DOIs
StatePublished - 2002

Fingerprint

Environmental Carcinogens
Nucleotides
DNA-Directed DNA Polymerase
Catalytic Domain
Guanine
DNA Adducts
Base Pairing
Carcinogens
Hydrogen
Bacteriophage T7
Proteins
Benzo(a)pyrene
DNA
Epoxy Compounds
Molecular Dynamics Simulation
Hydrogen Bonding
2'-deoxycytidine 5'-triphosphate
Mutation
deoxyguanosine triphosphate
thymidine 5'-triphosphate

Keywords

  • Carcinogen-DNA adduct
  • Carcinogen-induced mutagenicity
  • Environmental carcinogen
  • Nucleotide incorporation

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Toward understanding the mutagenicity of an environmental carcinogen: Structural insights into nucleotide incorporation preferences",
abstract = "Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N2-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be mis-replicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N2-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A > T ≥ G > C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N2-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N2-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N2-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N2-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N2-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N2-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N2-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N2-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N2-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.",
keywords = "Carcinogen-DNA adduct, Carcinogen-induced mutagenicity, Environmental carcinogen, Nucleotide incorporation",
author = "Perlow, {Rebecca A.} and Suse Broyde",
year = "2002",
doi = "10.1016/S0022-2836(02)00751-9",
language = "English (US)",
volume = "322",
pages = "291--309",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
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TY - JOUR

T1 - Toward understanding the mutagenicity of an environmental carcinogen

T2 - Structural insights into nucleotide incorporation preferences

AU - Perlow, Rebecca A.

AU - Broyde, Suse

PY - 2002

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N2 - Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N2-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be mis-replicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N2-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A > T ≥ G > C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N2-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N2-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N2-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N2-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N2-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N2-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N2-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N2-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N2-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.

AB - Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N2-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be mis-replicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N2-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A > T ≥ G > C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N2-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N2-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N2-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N2-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N2-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N2-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N2-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N2-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N2-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.

KW - Carcinogen-DNA adduct

KW - Carcinogen-induced mutagenicity

KW - Environmental carcinogen

KW - Nucleotide incorporation

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