Energetic and Structural Bases of DNA Overstretching Transition and its Cooperativity

By using a dual laser optical tweezers with a fast force feedback, we were able to record the exponential elongation following force steps imposed on Δ-phage ds-DNA molecule and on two ∼3000 bp segments of the molecule, with either high (59%) or low (46%) CG content. The rate of elongation (r) following a 2pN step imposed on the whole molecule in the region of the overstretching transition varies with the force in a U shaped way, while its corresponding elongation varies in a reverse-U shaped way (Bianco et al., Biophys. J. 101, 866-874, 2011). The minimum of the r - force relation (rmin) and the corresponding maximum elongation (ΔLe) do not change significantly with temperature in the range 25-10°C (mean values 4.9 ± 0.2 s-1 and 3.9 ± 0.2 μm respectively) and are shifted progressively to higher forces at lower temperature. The load- and temperature-dependence of the elongation rate supports the two state (B-S) nature of the transition, yielding a cooperativity of 22 bp and revealing the absence of an enthalpic contribution to the transition free energy barrier.At room temperature the AT rich segment shows large hysteresis on relaxation, implying a predominance of melting on overstretching, while the CG rich segment does not show hysteresis. At temperature below 10°C hysteresis completely disappears for both segments. rmin and ΔLe are 3.9 ±1.4 s-1 and 0.3 ± 0.1 nm for the CG and 4.3 ± 1.3 s-1 and 0.3 ± 0.1 nm for the AT. Fitting the data with the two state reaction model shows a cooperativity of ∼ 15 and 32 for the AT and CG respectively, which correlates with the average distance between groups of more than four consecutive A or T bases.Supported by IIT-SEED (Italy).