Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous DNA. Footprinting with methidiumpropyl-EDTA•iron(II)

Abstract

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE•Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with ^(32)P at the 3' end. MPE•Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg^(2+). The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg^(2+) appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG•dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE•Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg^(2+), the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA > CCG, GCC > CGA, CCT > CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.

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