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 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.
Thiyagarajan
M M
MM
Department of Pharmacology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
Waldman
S A
SA
Noè
M
M
Kmiec
E B
EB
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D013379:Substrate Specificity; D014588:Ustilago / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I