DNA–Protein Cross-Link Formation and Cellular Toxicity by Chimeric Bis-Electrophiles

DNA-protein cross-links (DPCs) and DNA-DNA interstrand cross-links (ICLs) strongly block genetic expression. Chemical agents that preferentially produce one of these DNA damage types over the other are potentially useful cellular probes and pharmacological agents but are uncommon. A chimeric alkylating agent (MEBAC) selectively forms DPCs over ICLs by reacting with the ε-amine of lysine. Here we determine that the majority of DPCs formed by MEBAC in NCPs are between dG and lysines in the histone amino terminal tails. We also compare the reactivity of MEBAC and an isomer (<i>m</i>-MEBAC). Model studies indicate that <i>m</i>-MEBAC reacts ∼50% more rapidly than MEBAC with a primary amine. DPC yields in nucleosome core particles (NCPs) from MEBAC and <i>m</i>-MEBAC are approximately equal at all but the highest concentration tested. Comparisons between the reactivity of MEBAC and a nitrogen mustard with NCPs and nuclear lysates illustrate the advantage of the former for generating DPCs. <i>m</i>-MEBAC and MEBAC cytotoxicity in human cells are comparable and are enhanced when proteasomal DPC repair is inhibited. These experiments validate the utility of this family of chimeric bis-electrophiles as chemical tools for forming DPCs in the test tube and in cells.