Figure 4

dsDNA is nicked by RepD only when it is subjected to negative supercoiling. Beads that were tethered to the surface by a single, 4-kb, dsDNA molecule were identified by the characteristic change in DNA length upon supercoiling (n_obs = ~70). The DNA molecules were then positively supercoiled by +20 turns before 100 nM RepD was added to the experimental flow-cell. All DNA molecules remained intact until they were subjected to small levels of negative supercoiling. After −4.5 ± 0.1 turns (±SEM) of negative supercoiling (σ = −1.2%), 50% of the DNA molecules with the wild type oriD sequence (circles) were nicked by RepD. The oriD mutant mut2/3 (squares), had the 50% nicking threshold at −5.6 ± 0.2 turns. Experiments were at F = 0.4 pN and 23 °C. The exact level of supercoiling for each DNA molecule was corrected for its initial starting offset due to thermal motion (see main text for details). At low levels of supercoiling, the elastic energy (Δwork) due to changes in DNA torque and secondary structure formation approximates to the function: \(\frac{C}{2{{\rm{l}}}_{{\rm{O}}}}[{(2{\rm{\pi }}{\rm{n}}+2{\rm{\pi }}h)}^{2}-{(2{\rm{\pi }}{\rm{n}})}^{2}]\) where C is DNA torsional stiffness (240 pN.nm².rad⁻¹ per unit length²⁷), l_o is the DNA length (here, 4000 bp * 0.34 nm/bp = 1360 nm), n, the number of supercoiling turns and h, the number of helical turns transferred from dsDNA backbone into hairpin structure. E_loop is the enthalpic energy cost of unstacking and unpairing bases in the DNA loop regions (see main text). The least-squares, fitted-lines are to the relationship: \(y={(1+ex{p}^{(\frac{-{E}_{loop}-{\rm{\Delta }}work}{{{\rm{k}}}_{{\rm{b}}}{\rm{T}}})})}^{-1}\) Fitting parameters were: wild type oriD: h = 0.91 turns and E_loop = 31 pN.nm; and mut2/3: h = 0.71 turns and E_loop = 29 pN.nm. The leftward shift of the mut2/3 oriD data compared to wild type is equivalent to an additional torsional energy requirement of ~34 pN.nm.