Mass spectrometry based approach to study the kinetics of O 6-alkylguanine DNA alkyltransferase-mediated repair of O 6-pyridyloxobutyl-2′-deoxyguanosine adducts in DNA

Delshanee Kotandeniya, Dan Murphy, Uthpala Seneviratne, Rebecca Guza, Anthony Pegg, Sreenivas Kanugula, Natalia Tretyakova

Research output: Contribution to journalArticle

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Abstract

O 6-POB-dG (O 6-[4-oxo-4-(3-pyridyl)but-1-yl] deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O 6-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O 6-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O 6-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O 6-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI +-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O 6-POB-dG adducts. In our approach, synthetic DNA duplexes containing O 6-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D 4-O 6-POB-dG internal standard and mild acid hydrolysis to release O 6-POB-guanine (O 6-POB-G) and D 4-O 6-POB-guanine (D 4-O 6-POB-G), samples are purified by solid phase extraction (SPE), and O 6-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI +-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O 6-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O 6-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI +-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O 6-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5′-AATAGTATCT[O 6-POB-G]GAGCC- 3′) opposite either C or T. Faster rates of alkyl transfer were observed when O 6-POB-dG was paired with T rather than with C (k = 1.74 × 10 6 M -1 s -1 vs 1.17 × 10 6 M -1 s -1).

Original languageEnglish (US)
Pages (from-to)1966-1975
Number of pages10
JournalChemical research in toxicology
Volume24
Issue number11
DOIs
StatePublished - Nov 21 2011

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O(6)-Methylguanine-DNA Methyltransferase
Deoxyguanosine
DNA Adducts
Mass spectrometry
Mass Spectrometry
Repair
Kinetics
DNA
Guanine
High Pressure Liquid Chromatography
Mutagenesis
DNA sequences
Nitrosamines
Proteins
Electrospray Ionization Mass Spectrometry
Solid Phase Extraction
Alkylation
Tandem Mass Spectrometry
Site-Directed Mutagenesis
DNA Replication

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Kotandeniya, Delshanee ; Murphy, Dan ; Seneviratne, Uthpala ; Guza, Rebecca ; Pegg, Anthony ; Kanugula, Sreenivas ; Tretyakova, Natalia. / Mass spectrometry based approach to study the kinetics of O 6-alkylguanine DNA alkyltransferase-mediated repair of O 6-pyridyloxobutyl-2′-deoxyguanosine adducts in DNA. In: Chemical research in toxicology. 2011 ; Vol. 24, No. 11. pp. 1966-1975.
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abstract = "O 6-POB-dG (O 6-[4-oxo-4-(3-pyridyl)but-1-yl] deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O 6-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O 6-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O 6-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O 6-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI +-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O 6-POB-dG adducts. In our approach, synthetic DNA duplexes containing O 6-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D 4-O 6-POB-dG internal standard and mild acid hydrolysis to release O 6-POB-guanine (O 6-POB-G) and D 4-O 6-POB-guanine (D 4-O 6-POB-G), samples are purified by solid phase extraction (SPE), and O 6-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI +-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O 6-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O 6-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI +-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O 6-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5′-AATAGTATCT[O 6-POB-G]GAGCC- 3′) opposite either C or T. Faster rates of alkyl transfer were observed when O 6-POB-dG was paired with T rather than with C (k = 1.74 × 10 6 M -1 s -1 vs 1.17 × 10 6 M -1 s -1).",
author = "Delshanee Kotandeniya and Dan Murphy and Uthpala Seneviratne and Rebecca Guza and Anthony Pegg and Sreenivas Kanugula and Natalia Tretyakova",
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Mass spectrometry based approach to study the kinetics of O 6-alkylguanine DNA alkyltransferase-mediated repair of O 6-pyridyloxobutyl-2′-deoxyguanosine adducts in DNA. / Kotandeniya, Delshanee; Murphy, Dan; Seneviratne, Uthpala; Guza, Rebecca; Pegg, Anthony; Kanugula, Sreenivas; Tretyakova, Natalia.

In: Chemical research in toxicology, Vol. 24, No. 11, 21.11.2011, p. 1966-1975.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mass spectrometry based approach to study the kinetics of O 6-alkylguanine DNA alkyltransferase-mediated repair of O 6-pyridyloxobutyl-2′-deoxyguanosine adducts in DNA

AU - Kotandeniya, Delshanee

AU - Murphy, Dan

AU - Seneviratne, Uthpala

AU - Guza, Rebecca

AU - Pegg, Anthony

AU - Kanugula, Sreenivas

AU - Tretyakova, Natalia

PY - 2011/11/21

Y1 - 2011/11/21

N2 - O 6-POB-dG (O 6-[4-oxo-4-(3-pyridyl)but-1-yl] deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O 6-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O 6-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O 6-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O 6-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI +-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O 6-POB-dG adducts. In our approach, synthetic DNA duplexes containing O 6-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D 4-O 6-POB-dG internal standard and mild acid hydrolysis to release O 6-POB-guanine (O 6-POB-G) and D 4-O 6-POB-guanine (D 4-O 6-POB-G), samples are purified by solid phase extraction (SPE), and O 6-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI +-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O 6-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O 6-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI +-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O 6-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5′-AATAGTATCT[O 6-POB-G]GAGCC- 3′) opposite either C or T. Faster rates of alkyl transfer were observed when O 6-POB-dG was paired with T rather than with C (k = 1.74 × 10 6 M -1 s -1 vs 1.17 × 10 6 M -1 s -1).

AB - O 6-POB-dG (O 6-[4-oxo-4-(3-pyridyl)but-1-yl] deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O 6-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O 6-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O 6-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O 6-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI +-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O 6-POB-dG adducts. In our approach, synthetic DNA duplexes containing O 6-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D 4-O 6-POB-dG internal standard and mild acid hydrolysis to release O 6-POB-guanine (O 6-POB-G) and D 4-O 6-POB-guanine (D 4-O 6-POB-G), samples are purified by solid phase extraction (SPE), and O 6-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI +-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O 6-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O 6-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI +-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O 6-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5′-AATAGTATCT[O 6-POB-G]GAGCC- 3′) opposite either C or T. Faster rates of alkyl transfer were observed when O 6-POB-dG was paired with T rather than with C (k = 1.74 × 10 6 M -1 s -1 vs 1.17 × 10 6 M -1 s -1).

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