Determination of the Free-Energy Change for Repair of a DNA Phosphodiester Bond

Abstract

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 i , was quantified. At 25°C, pH 7, the equilibrium constant (K eq ) for the ligation reaction is 3.89 ؋ 10 4 M. This value corresponds to a standard free energy (⌬G°) of ؊6.3 kcal mol ؊1 . By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP i , ⌬G° for the hydrolysis of a DNA phosphodiester bond is ؊5.3 kcal mol ؊1 .The repair of a single-strand break in DNA involves synthesis of a phosphodiester bond by condensation of a 5Ј-phosphoryl group on one nucleotide with a 3Ј-hydroxyl group on an adjacent nucleotide. DNA ligases catalyze this reaction by coupling condensation to cleavage of a phosphoanhydride bond from either NAD ϩ or ATP (1). The overall reaction favors phosphodiester bond formation. However, a quantitative value for this energy change has never been reported, nor has the standard free energy for the hydrolysis of a phosphodiester bond in DNA been determined. T4 DNA ligase, as well as ligases from eukaryotic and some prokaryotic cells, uses ATP as a co-substrate to catalyze DNA repair (2). The DNA ligase catalyzed reaction is reversible, as first demonstrated for Escherichia coli DNA ligase by conversion of a supercoiled circular DNA to a relaxed circular form (3). The reaction scheme for ATP-dependant DNA ligases is illustrated in Scheme 1.T4 DNA ligase, acting on a circular DNA template, was used in a DNA nicking assay to calculate an equilibrium constant (K eq ) for the ligation reaction. From this value, the corresponding standard free energies (⌬G°Ј) of both the ligation and phosphodiester bond hydrolysis reactions were determined. EXPERIMENTAL PROCEDURES T4 DNA ligase was purchased from New England Biolabs, Inc. and had a specific activity of 10 6 NEB units/mg. ATP was from Roche Molecular Biochemicals, and AMP was from Sigma. The concentrations of the adenine nucleotides were determined spectrophotometrically using ⑀ 259 ϭ 1.54 ϫ 10 4 . Na 4 P 2 O 7 -10 H 2 O was from Fisher Scientific. Ethidium bromide was from Sigma. All other reagents were ACS certified or better.The two circular plasmid DNAs used were as follows: the 3. et al. (6). This method involved concentrating the supercoiled DNA by centrifugation through a cesium chloride density gradient containing ethidium bromide. Residual ethidium bromide was subsequently removed from the DNA preparation by extraction with isobutanol, and the concentration of the DNA was determined as described by Dillon et al. (6). Linear DNA was formed by digestion with HindIII (New England Biolabs). Ligation reactions were performed in a sta...

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