DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells
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- 作者：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
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：May 7, 2013
- 年：2013
- 卷：52
- 期：18
- 页码：3171-3181
- 全文大小：455K
- 年卷期：v.52,no.18(May 7, 2013)
- ISSN：1520-4995
文摘
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鈥損rotein cross-links (DPCs). Interstrand DNA鈥揇NA 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鈥揇NA 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.