Repair kinetics of trans-4-Hydroxynonenal-induced cyclic 1,N 2-propanodeoxyguanine DNA adducts by human cell nuclear extracts

Sujata Choudhury, Jishen Pan, Shantu Amin, Fung Lung Chung, Rabindra Roy

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of ω-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N2-γ-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [32P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were ∼1200/106 dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of α[32P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [32P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 °C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.

Original languageEnglish (US)
Pages (from-to)7514-7521
Number of pages8
JournalBiochemistry
Volume43
Issue number23
DOIs
StatePublished - Jun 15 2004

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DNA Adducts
Cell Extracts
Repair
Cells
Kinetics
DNA
Substrates
4-hydroxy-2-nonenal
HeLa Cells
DNA Repair
Isomers
Xeroderma Pigmentosum Group A Protein
Plasmids
Nucleotides
High Pressure Liquid Chromatography
Hepatolenticular Degeneration
Deoxyguanosine
Unsaturated Fatty Acids
Liver
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Choudhury, Sujata ; Pan, Jishen ; Amin, Shantu ; Chung, Fung Lung ; Roy, Rabindra. / Repair kinetics of trans-4-Hydroxynonenal-induced cyclic 1,N 2-propanodeoxyguanine DNA adducts by human cell nuclear extracts. In: Biochemistry. 2004 ; Vol. 43, No. 23. pp. 7514-7521.
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abstract = "trans-4-Hydroxynonenal (HNE) is a major peroxidation product of ω-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N2-γ-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [32P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were ∼1200/106 dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of α[32P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [32P]-postlabeling/HPLC method, showing that 50-60{\%} of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 °C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.",
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Repair kinetics of trans-4-Hydroxynonenal-induced cyclic 1,N 2-propanodeoxyguanine DNA adducts by human cell nuclear extracts. / Choudhury, Sujata; Pan, Jishen; Amin, Shantu; Chung, Fung Lung; Roy, Rabindra.

In: Biochemistry, Vol. 43, No. 23, 15.06.2004, p. 7514-7521.

Research output: Contribution to journalArticle

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T1 - Repair kinetics of trans-4-Hydroxynonenal-induced cyclic 1,N 2-propanodeoxyguanine DNA adducts by human cell nuclear extracts

AU - Choudhury, Sujata

AU - Pan, Jishen

AU - Amin, Shantu

AU - Chung, Fung Lung

AU - Roy, Rabindra

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Y1 - 2004/6/15

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AB - trans-4-Hydroxynonenal (HNE) is a major peroxidation product of ω-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N2-γ-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [32P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were ∼1200/106 dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of α[32P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [32P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 °C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.

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