The pathway for base pair opening within a B-DNA duplex is investigated by theoretical molecular modeling. The results show that the disruption of a single base pair is energetically compatible with the deductions made from hydrogen exchange measurements. In addition, it is found that the opening process is greatly facilitated by DNA bending and that, conversely, once a base pair is disrupted, DNA can bend very easily. It appears that the energetic coupling between these two processes may play an important role in many biological reactions involving nucleic acid distortion. Despite the accumulation of a wealth of information leading to a better knowledge of the chemical mechanism and of the corresponding time range (1-4), the conformational and energetic details of the pathway for opening and the effect of changes in the tertiary structure of DNA remain unclear. Most notably, the mechanism by which a given base pair manages to accumulate enough energy to spontaneously open and disrupt its hydrogen bonds is still fully unknown. These questions are addressed here by theoretical calculations on a B-DNA oligomer. Using the JUMNA (junction minimization of nucleic acids) molecular modeling program (5), we study the energetics and the conformational route for opening one base within a short DNA fragment and then demonstrate how base pair opening and bending of the DNA double helix are related to one another. METHODSAll of the conformational energy calculations presently described were carried out by using the FLEX parameterization (6, 7) developed over several years in our laboratory and already exploited in a large number of studies of nucleic acids and other biological molecules (see, for example, refs. 8-10). The calculations described here were performed under dielectric damping conditions corresponding to aqueous solution (S = 0.16, D = 78). The next three energy terms represent dispersion-repulsion interactions calculated with a 6-12 dependence using, in part, the parameter set developed by Zhurkin et al. (14). Hydrogen bonds (HB) are dealt with by the latter two of these terms, which take into account their angular dependence. All of these terms are summed over pairs of atoms separated by at least three chemical bonds.The last two terms represent the distortion energy associated with torsion angles T, (including anomeric effects) and valence angles a,-To study the base-opening or bending processes within a DNA fragment it is necessary to have control over the helicoidal parameters describing the position of the bases in space as well as parameters indicating the path of the helical axis so that a given "open" or "bent" state can be maintained while energy optimizing the rest of the DNA conformation. This possibility, which is unavailable in normal molecular mechanics procedures, can be achieved with the recently developed JUMNA procedure (5).The aim of the JUMNA approach is to combine the control over the helicoidal parameters of DNA with the rapidity and ease of treating internal conformation changes, i...

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The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature t m = 43 8C and 41 8C, denaturation enthalpies DH = 46 kcal´mol ±1 ). The thermal melting of protein A-B presents two nearly identical transitions (t m = 40 8C and 41 8C, DH = 44 kcal´mol ±1 and 46 kcal´mol ±1 , respectively). We conclude that the two domains A and B within protein A-B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of t m (38 8C and 55 8C) and corresponding values of DH around 40 kcal´mol ±1 . We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other. At 37 8C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal´mol ±1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy.