Visualizing single DNA-bound proteins using DNA as a scanning probe.
Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.
Noom
Maarten C
MC
Physics of Complex Systems, Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
van den Broek
Bram
B
van Mameren
Joost
J
Wuite
Gijs J L
GJ
D001665:Binding Sites; D015342:DNA Probes / Q000737:chemistry* / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D020527:Microscopy, Scanning Probe / Q000379:methods*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation
D015342:DNA Probes; D004268:DNA-Binding Proteins