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Published on: November 10, 2016
Binding characteristics of Ustilago maydis topoisomerase I to DNA containing secondary structures
M M Thiyagarajan1, S A Waldman, M Noè
1Department of Pharmacology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
Abstract:
The binding affinity of purified native Ustilago maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary structures was determined by gel shift and equilibrium binding analysis. Topoisomerase I exhibited cooperativity in binding to DNA regardless of the substrate structure. Further analysis demonstrated that cruciform DNA 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 topoisomerase I for cruciform was found to be an order of magnitude higher affinity than for any of the other substrates. The high affinity of topoisomerase I for cruciform and specificity of topoisomerase I-cruciform structure interaction were confirmed by competition experiments. These studies demonstrate the high affinity of topoisomerase I for cruciform structure.
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