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DNA-Protein Cross-Link Formation and Cellular Toxicity by Chimeric Bis-Electrophiles
Hanrui Yu1, Xuanhe Jiang1, Marc M Greenberg1
1Johns Hopkins University, Department of Chemistry, 3400 N. Charles St., Baltimore, Maryland 21218, United States.
Abstract:
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 (m-MEBAC). Model studies indicate that m-MEBAC reacts ∼50% more rapidly than MEBAC with a primary amine. DPC yields in nucleosome core particles (NCPs) from MEBAC and m-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. m-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.
Insights
Researchers developed a new chemical agent, MEBAC, that selectively creates DNA-protein cross-links (DPCs), which are crucial for studying DNA damage and developing new therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- DNA-protein cross-links (DPCs) and DNA-DNA interstrand cross-links (ICLs) impede gene expression.
- Agents that selectively induce DPCs or ICLs are valuable research tools and potential therapeutics but are rare.
Purpose of the Study:
- To investigate the specificity and utility of a chimeric alkylating agent, MEBAC, for forming DPCs.
- To compare the reactivity and efficacy of MEBAC with its isomer, m-MEBAC, and a nitrogen mustard.
Main Methods:
- Characterization of DPCs formed by MEBAC in nucleosome core particles (NCPs).
- Comparative reactivity studies of MEBAC and m-MEBAC with primary amines and NCPs.
- Cytotoxicity assays in human cells, assessing the role of proteasomal DPC repair.
Main Results:
- MEBAC predominantly forms DPCs between deoxyguanosine (dG) and lysines in histone tails within NCPs.
- m-MEBAC exhibits slightly higher reactivity with primary amines but similar DPC yields to MEBAC in NCPs.
- MEBAC demonstrates an advantage over nitrogen mustard in generating DPCs in NCPs and nuclear lysates.
- MEBAC and m-MEBAC show comparable cytotoxicity, enhanced by inhibiting proteasomal DPC repair.
Conclusions:
- MEBAC is a selective chemical tool for generating DPCs in vitro and in cellular systems.
- The chimeric bis-electrophile family, including MEBAC and m-MEBAC, offers a promising approach for DPC induction.
- Understanding DPC formation and repair is critical for developing targeted cancer therapies.
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