Subramani, R.; Juul, S.; Rotaru, A.; Andersen, F. F.; Gothelf, K. V.; Mamdouh, W.; Besenbacher, F.; Dong, M.; Knudsen, B. R.
ACS Nano 2010, 4, 5969–5977, doi: 10.1021/nn101662a
Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Nordre Ringgade 1, DK-8000 Aarhus C, Denmark
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.