Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation.

During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.