Thermodynamic, Kinetic, and Structural Basis for Recognition and Repair of 8-Oxoguanine in DNA by Fpg Protein from Escherichia coli

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Abstract
X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides ∼7 orders of magnitude of affinity (ΔG° ≈ −9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (ΔG° ≈ −0.90 kcal/mol) together with other specific interactions is G° ≈ −2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the kcat term; the rate is increased by 6−8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.