Real-Time Detection of Cruciform Extrusion by Single-Molecule DNA Nanomanipulation

Abstract

Cruciform extrusion in dsDNA can occur when a DNA palindrome is subjected to physiological levels of negative supercoiling. Here we use single-DNA nanomanipulation to explore the kinetic and structural properties of cruciform extrusion induced by negative supercoiling. Cruciform extrusion appears as an abrupt increase in the extension of negatively supercoiled DNA, and the amplitude of the change in extension is proportional to the number of bases in the cruciform. The kinetics of this two-state system, B-DNA and cruciform DNA, can be tuned by negative supercoiling which destabilizes the former and stabilizes the latter. The rate of extrusion is controlled by the size of the apical loop, decreasing as the loop size is increased from 5 to 8 bases. Cruciform rewinding is controlled by features in the stem. Perfect cruciforms will tend to extrude irreversibly, whereas shortening and addition of imperfections to a stem can render extrusion reversible. From measurements of the effect of torque on extrusion/rewinding kinetics we propose that in the transition state to cruciform extrusion the palindrome is unwound in the unpaired loop region of the cruciform. These results provide insight into the mechanism of cruciform extrusion and help to understand the potential role of these structures in processes of genomic instability as well as those underpinning the synthesis of non-coding RNAs.