A novel secondary DNA binding site in human topoisomerase I unravelled by using a 2D DNA origami platform.
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.
Subramani
Ramesh
R
Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Nordre Ringgade 1, DK-8000 Aarhus C, Denmark.
Juul
Sissel
S
Rotaru
Alexandru
A
Andersen
Felicie F
FF
Gothelf
Kurt V
KV
Mamdouh
Wael
W
Besenbacher
Flemming
F
Dong
Mingdong
M
Knudsen
Birgitta R
BR
D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005782:Gels; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D008968:Molecular Conformation; D036103:Nanotechnology / Q000379:methods; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D004270:DNA, Circular; D005782:Gels; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D004264:DNA Topoisomerases, Type I