DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

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. Lower 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.

Covalent DNA–Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells

“…45 Furthermore, proteins functionalized with DNA-reactive 2-hydroxy-3,4-epoxybutyl groups induced cell death and mutations in human cells. 46 …”

Synthesis of Sequence-Specific DNA–Protein Conjugates via a Reductive Amination Strategy

“…The availability of hydrolytically stable model DPCs substrates will, for the first time, enable structural and biological evaluation of these super-bulky lesions. Based on our recent studies with DNA-reactive protein reagents that specifically induce DPCs in cells, 20 we hypothesize that spontaneous and xenobiotic-induced DPCs, if not repaired, compromise the efficiency and the accuracy of DNA replication and are responsible for a major portion of the toxicity and mutagenicity induced by bis- alkylating agents, UV light, reactive oxygen species, and γ-radiation.…”

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…Because of their unusual size, significant heterogeneity, and the ability to disrupt normal DNA-protein interactions (2-6), DPCs are hypothesized to block DNA replication, transcription, and repair and to induce cytotoxic and mutagenic effects (7)(8)(9). However, the exact biological consequences of these inherently complex DNA lesions are not fully understood due to their structural diversity and the difficulty in generating site-specific DPCs for biological studies.…”