Increased flexibility enhances misincorporation: Temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase

Rebecca A. Perlow-Poehnelt, Ilya Likhterov, Lihua Wang, David A. Scicchitano, Nicholas E. Geacintov, Suse Broyde

Research output: Contribution to journalArticle

Abstract

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N 2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55°C and results compared with an earlier study at 37°C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55°C compared with 37°C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55°C compared with 37°C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the k cat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

Original languageEnglish (US)
Pages (from-to)1397-1408
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number2
DOIs
StatePublished - Jan 12 2007

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DNA Adducts
DNA-Directed DNA Polymerase
Carcinogens
Thermal effects
Nucleotides
Temperature
Molecular Dynamics Simulation
Conformations
Molecular dynamics
Sulfolobus solfataricus
Molecular modeling
Benzo(a)pyrene
Computer simulation
Guanine
Metabolites
Cats
Kinetics
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Increased flexibility enhances misincorporation: Temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase",
abstract = "The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N 2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55°C and results compared with an earlier study at 37°C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55°C compared with 37°C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55°C compared with 37°C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the k cat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.",
author = "Perlow-Poehnelt, {Rebecca A.} and Ilya Likhterov and Lihua Wang and Scicchitano, {David A.} and Geacintov, {Nicholas E.} and Suse Broyde",
year = "2007",
month = "1",
day = "12",
doi = "10.1074/jbc.M606769200",
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journal = "Journal of Biological Chemistry",
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T1 - Increased flexibility enhances misincorporation

T2 - Temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase

AU - Perlow-Poehnelt, Rebecca A.

AU - Likhterov, Ilya

AU - Wang, Lihua

AU - Scicchitano, David A.

AU - Geacintov, Nicholas E.

AU - Broyde, Suse

PY - 2007/1/12

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N2 - The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N 2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55°C and results compared with an earlier study at 37°C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55°C compared with 37°C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55°C compared with 37°C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the k cat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

AB - The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N 2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55°C and results compared with an earlier study at 37°C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55°C compared with 37°C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55°C compared with 37°C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the k cat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

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