Determining the Elasticity of Short DNA Fragments using Optical Tweezers and Protein-Mediated DNA Loop Formation Assays

Abstract

different locations along the genome and changes during the cell cycle, little is known about how localized changes in DNA supercoiling, under enzymegenerated DNA tension, perturb critical protein-DNA interactions. Here we investigated how DNA supercoiling affects stability of the lambda repressormediated DNA loop that acts as a genetic switch between lysogeny (quiescence) and lysis (virulence). We performed single molecule magnetic tweezers measurements to record lambda repressor-mediated DNA loop formation and breakdown and to measure the stability of the loop as a function of negative supercoiling, loop size and DNA tension at physiological repressor concentration. The level of negative supercoiling required for loop formation increases with loop size and that, in general, negative supercoiling stabilizes the loop. Since genomic supercoiling depends on the energy level of the cell which is tightly associated with its health status, we propose that the switch to lysis is favored by the destabilization of the lambda repressor-mediated loop that follows loss of DNA supercoiling in suffering cells. 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 D-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 (DLe) do not change significantly with temperature in the range 25-10 C (mean values 4.9 5 0.2 s-1 and 3.9 5 0.2 mm 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 DLe are 3.9 51.4 s-1 and 0.3 5 0.1 nm for the CG and 4.3 5 1.3 s-1 and 0.3 5 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.