Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion.
Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB-DNA complexes has suggested that the enzyme promotes long-range DNA-DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.
Moreno-Herrero
Fernando
F
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Holtzer
Laurent
L
Koster
Daniel A
DA
Shuman
Stewart
S
Dekker
Cees
C
Dekker
Nynke H
NH
D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D014616:Vaccinia virus / Q000201:enzymology*
D004247:DNA; D004264:DNA Topoisomerases, Type I