A Nuclear Magnetic Resonance Investigation of the Energetics of Basepair Opening Pathways in DNA

Abstract

The opening of basepairs plays a key role in DNA replication and transcription, and in the action of DNA repair and modification enzymes. In this article, we have used proton exchange to define the energetics of the pathways for basepair opening in two DNA 17-mer duplexes. The rates of exchange of imino protons with solvent protons were measured by NMR spectroscopy for each DNA duplex, as a function of the concentration of exchange catalyst and of temperature. The measurements provided the rates and the equilibrium constants of the opening reactions for individual basepairs at different temperatures. These temperature dependences were used to calculate the enthalpies and the free energies of the barrier to opening and of the open state for each basepair. The results reveal the existence of three distinct patterns of enthalpy changes in the opening reactions. The patterns differ from each other in the location of the kinetic opening barrier relative to the open state. Neighboring bases, which are one or more positions removed from the opening basepair, influence the enthalpic pattern of the opening pathway. The free energies of the opening barriers are found to be linearly related to the free energies of the open state. This correlation is analyzed in terms of rate-equilibrium free energy relationships previously observed in other systems, and suggests that the transition state in the opening reaction is closer to the native closed state of the basepair than to its open state.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

“…Similarly, for bend Ϸ 12.8 ms at 20°C, we get ⌬G bubble † in the range of 6.2-8.3 kcal͞mol. These estimates for the free energy cost of opening a bubble inside DNA correlate very well with the range of equilibrium free energy changes, of Ϸ5.6 -9.2 kcal͞mol, for single base pair opening at 22°C, obtained from imino-proton exchange measurements (37), and lend further support to the suggestion that that the bending times observed in our measurements correspond to the time scale for disruption of a single internal base pair in B-DNA.…”

“…In addition, the slope of the Arhennius plot in Fig. 5c for DNA bending is within the range of activation energies determined for single base pair opening (37). Hence, it is plausible that opening a single A:T base pair is sufficient to nucleate DNA bending.…”

“…Thus, the opening of bubbles in the DNA duplex, when it is tightly wrapped around IHF, is much slower than the DNA bending times observed in our kinetics measurements, and also much slower than the opening͞closing times observed at a single base pair level for uncomplexed B-DNA (36)(37)(38).…”

“…Recent determination of base pair lifetimes, monitored by NMR measurement of imino proton exchange kinetics (36,37), have shown that single A:T base pair opening rates in B-DNA bracket the measured DNA bending rate from 10°C to 30°C (Fig. 5c).…”

Entropic penalties in circular DNA assembly

“…Also the values of entropy per base pair are larger than those obtained by the previous method and S1 − S2, at the values of helical repeat which maximize S0 and S1. [19,56], although they remain smaller by a factor five than the experimental estimates [57][58][59]. Nevertheless, this discrepancy can be at least partly reconciled by further tuning the model parameters and, specifically, by softening the hydrogen bond potential which, in turn, yields higher entropy values.…”