Highly Preferential Linker Histone Binding to Actinomycin D-Treated DNA

Abstract

ABSTRACTElectromobility-shift assay (EMSA) was applied for studying the competition between the anticancer antibiotic actinomycin D (ACTD) and linker histones H1 and H5 for binding to DNA. ACTD is widely used in the treatment of some malignancies. Its binding to DNA occurs in two steps: initial intercalation between guanine and cytosine bases followed by invading the minor groove of the polynucleotide chain. As a consequence of this structural perturbation of DNA, a strong interference with the binding of important regulatory and functional proteins to DNA may occur. Here we present our recent experimental data obtained in a drug-competition assay between ACTD and linker histones H1 and H5 under the following conditions: i) simultaneous incubation of DNA with the antibiotic and histone; ii) preincubation of DNA with ACTD and subsequent addition of the histone; and iii) preincubation of DNA with histone and subsequent addition of the drug. Surprisingly, linker histones bound more effectively to ACTD-pretreated DNA than to the untreated control. The results obtained with plasmid DNA were confirmed using a series of various native and synthetic DNA fragments bearing different number of antibiotic targets. The same results were obtained with the globular domain of H5, the portion of the linker histone suggested to be most responsible for DNA binding. The data with another nuclear protein with similar DNA-binding properties as linker histones—HMGB1—showed strongly reduced contacts with ACTD-treated DNA compared with the untreated.