Determination of the free-energy change for repair of a DNA phosphodiester bond

The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases, Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP,, was quantified. At 25 degrees C, pH 7, the equilibrium constant (K-eq) for the ligation reaction is 3.89 x 10(4) M. This value corresponds to a standard free energy (Delta G degrees') of -6.3 kcal mol(-1). By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PPi, Delta G degrees' for the hydrolysis of a DNA phosphodiester bond is -5.3 kcal mol(-1).