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A Novel Secondary DNA Binding Site in Human Topoisomerase I Unravelled by using a 2D DNA Origami Platform

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journal contribution
posted on 26.10.2010, 00:00 by Ramesh Subramani, Sissel Juul, Alexandru Rotaru, Felicie F. Andersen, Kurt V. Gothelf, Wael Mamdouh, Flemming Besenbacher, Mingdong Dong, Birgitta R. Knudsen
The biologically and clinically important nuclear enzyme human topoisomerase I relaxes both positively and negatively supercoiled DNA and binds consequently DNA with supercoils of positive or negative sign with a strong preference over relaxed DNA. One scheme to explain this preference relies on the existence of a secondary DNA binding site in the enzyme facilitating binding to DNA nodes characteristic for plectonemic DNA. Here we demonstrate the ability of human topoisomerase I to induce formation of DNA synapses at protein containing nodes or filaments using atomic force microscopy imaging. By means of a two-dimensional (2D) DNA origami platform, we monitor the interactions between a single human topoisomerase I covalently bound to one DNA fragment and a second DNA fragment protruding from the DNA origami. This novel single molecule origami-based detection scheme provides direct evidence for the existence of a secondary DNA interaction site in human topoisomerase I and lends further credence to the theory of two distinct DNA interaction sites in human topoisomerase I, possibly facilitating binding to DNA nodes characteristic for plectonemic supercoils.