BINDING CHARACTERISTICS OF USTILAGO-MAYDIS TOPOISOMERASE-I TO DNA CONTAINING SECONDARY STRUCTURES

The binding affinity of purified native Ustilago maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary struct ures was determined by gel shift and equilibrium binding analysis. Top oisomerase I exhibited cooperativity in binding to DNA regardless of t he substrate structure. Further analysis demonstrated that cruciform D NA has two populations of binding sites for topoisomerase I while the other substrates (single-stranded DNA, DNA molecules containing six or one mismatched base pairs, hairpin, and fully homologous duplex DNA) have a single population of binding sites. The affinity of topoisomera se I for cruciform was found to be an order of magnitude higher affini ty than for any of the other substrates. The high affinity of topoisom erase I for cruciform and specificity of topoisomerase I-cruciform str ucture interaction were confirmed by competition experiments. These st udies demonstrate the high affinity of topoisomerase I for cruciform s tructure.