DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells

Natalia Y. Tretyakova, Erin D. Michaelson-Richie, Teshome B. Gherezghiher, Jamie Kurtz, Xun Ming, Susith Wickramaratne, Melissa Campion, Sreenivas Kanugula, Anthony E. Pegg, Colin Campbell

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O6-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide- functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

Original languageEnglish (US)
Pages (from-to)3171-3181
Number of pages11
JournalBiochemistry
Volume52
Issue number18
DOIs
StatePublished - May 7 2013

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Mutagenesis
Cell death
Cell Death
Cells
DNA
Proteins
Toxicity
Hypoxanthine Phosphoribosyltransferase
Electroporation
Alkylating Agents
Epoxy Compounds
Cytotoxins
link protein
Biomolecules
Cell Nucleus
Transcription

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Tretyakova, N. Y., Michaelson-Richie, E. D., Gherezghiher, T. B., Kurtz, J., Ming, X., Wickramaratne, S., ... Campbell, C. (2013). DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. Biochemistry, 52(18), 3171-3181. https://doi.org/10.1021/bi400273m
Tretyakova, Natalia Y. ; Michaelson-Richie, Erin D. ; Gherezghiher, Teshome B. ; Kurtz, Jamie ; Ming, Xun ; Wickramaratne, Susith ; Campion, Melissa ; Kanugula, Sreenivas ; Pegg, Anthony E. ; Campbell, Colin. / DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. In: Biochemistry. 2013 ; Vol. 52, No. 18. pp. 3171-3181.
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Tretyakova, NY, Michaelson-Richie, ED, Gherezghiher, TB, Kurtz, J, Ming, X, Wickramaratne, S, Campion, M, Kanugula, S, Pegg, AE & Campbell, C 2013, 'DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells', Biochemistry, vol. 52, no. 18, pp. 3171-3181. https://doi.org/10.1021/bi400273m

DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. / Tretyakova, Natalia Y.; Michaelson-Richie, Erin D.; Gherezghiher, Teshome B.; Kurtz, Jamie; Ming, Xun; Wickramaratne, Susith; Campion, Melissa; Kanugula, Sreenivas; Pegg, Anthony E.; Campbell, Colin.

In: Biochemistry, Vol. 52, No. 18, 07.05.2013, p. 3171-3181.

Research output: Contribution to journalArticle

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AU - Tretyakova, Natalia Y.

AU - Michaelson-Richie, Erin D.

AU - Gherezghiher, Teshome B.

AU - Kurtz, Jamie

AU - Ming, Xun

AU - Wickramaratne, Susith

AU - Campion, Melissa

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AU - Pegg, Anthony E.

AU - Campbell, Colin

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Tretyakova NY, Michaelson-Richie ED, Gherezghiher TB, Kurtz J, Ming X, Wickramaratne S et al. DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. Biochemistry. 2013 May 7;52(18):3171-3181. https://doi.org/10.1021/bi400273m