DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells
DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells
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DOI：
10.1021/bi400273m
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发表时间：
2013-05-07
期刊：
影响因子：
2.9
通讯作者：
Campbell, Colin
中科院分区：
文献类型：
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作者：
Tretyakova, Natalia Y.;Michaelson-Richie, Erin D.;Campbell, Colin
尽管具有两个亲电反应基团的细胞毒性烷化剂被认为通过交联细胞生物分子起作用，但其确切的作用机制尚未建立。在细胞中，这些化合物形成DNA损伤的混合物，包括核碱基单加合物、链间和链内交联以及DNA蛋白质交联（DPC）。链间DNA DNA交联通过阻止DNA链分离来阻断复制和转录，从而导致毒性和诱变。相反，药物诱导的DPC的潜在贡献知之甚少。为了深入了解DPC形成的生物学后果，我们产生了DNA反应蛋白试剂，并在哺乳动物细胞中检测了它们的毒性和致突变性。重组人O-6-烷基鸟嘌呤DNA烷基转移酶（AGT）蛋白或其变体（C145 A和K125 L）用1，2，3，4-二环氧丁烷处理以产生在半胱氨酸残基上含有2-羟基-3，4-环氧丁基的蛋白。凝胶位移和质谱实验证实，环氧官能化AGT蛋白形成共价DPC，但没有其他类型的核碱基损伤时，与双链体DNA孵育。通过电穿孔将纯化的AGT单环氧化物引入哺乳动物细胞中产生AGT-DNA交联，并诱导细胞死亡和次黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因突变。当使用不能在细胞核中积累的AGT变体（K125 L）产生的蛋白单环氧化物时，观察到较少数量的DPC病变和降低的细胞死亡水平，这表明核DNA损伤是毒性所必需的。总之，这些结果表明AGT蛋白单环氧化物在染色体DNA内产生细胞毒性和致突变DPC损伤。更一般地说，这些数据表明，共价DPC病变有助于双亲电体的细胞毒性和致突变作用。
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 O-6-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.