Energetic Contributions of Plectoneme Tips and Tails

Abstract

Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme end-loops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails.