DNA-protein cross-linking by bifunctional DNA alkylating agents.

Abstract

Many common DNA alkylating agents, such as environmental toxins and chemotherapeutic drugs, are bis-electrophiles capable of covalently cross-linking cellular biomolecules. While DNA-DNA cross-linking by such compounds is well-characterized, the identities and the biological effects of the corresponding DNA-protein cross-links (DPCs) are poorly understood. Furthermore, because bis-electrophiles produce DNA-DNA cross-links and DNA monoadducts in addition to DPCs, it is difficult to establish the biological outcomes specifically resulting from DPC lesions. The purpose of the present work was to characterize DNA-protein cross-linking by two bis-electrophiles, 1,2,3,4-diepoxybutane (DEB) and bis(2-chloroethyl)methylamine (mechlorethamine), and to evaluate the ability of DPCs to induce cytotoxic and mutagenic effects. Mass spectrometry-based proteomics and immunological detection methods identified 41 proteins participating in DPC formation in the presence of DEB in nuclear protein extracts prepared from human cervical carcinoma (HeLa) cells, and 38 proteins which formed DPCs to the chromosomal DNA of human fibrosarcoma (HT1080) cells treated with mechlorethamine. Relative to their cellular abundance, a disproportionately high number of the proteins involved in DPC formation were nuclear proteins with known nucleic acid-binding capabilities which participate in cellular processes such as transcriptional regulation and DNA repair. HPLC-ESI+-MS/MS analysis of total proteolytic digests of DPCs revealed the chemical structures of the cross-links produced by DEB and mechlorethamine to be 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) and N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA), respectively. In order to analyze the biological consequences of DPC lesions, we selectively induced DPCs in mammalian cell cultures by electroporating them in the presence of epoxide-containing protein reagents. Significant levels of cell death and mutations were observed, suggesting that DPC lesions contribute to the biological effects of bis-electrophiles.