Ultraviolet-B-Induced Damage to Escherichia coli Fpg Protein

Abstract

Abstract— We investigated the effect of UVB light (290 ≤δ≤ 320 nm) on the structure and enzymatic activities of Escherichia coli Fpg protein (2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine-DNA glycosylase), a DNA repair enzyme containing a zinc finger motif and five chromophoric Trp residues. Irradiation with UVB light of air-saturated pH 7.4 buffered aqueous solutions of Fpg induces the formation of polymers as shown by sodium dodecyl sulfate polyaerylamide gel electrophoretic analysis. In argon-saturated solutions, polymer formation produces a precipitate. The polymerization quantum yield is 0.07 ± 0.01 and 0.15 ± 0.02 in air-and argon-saturated solutions, respectively. In the polymerized Fpg protein, second-derivative absorption spectroscopy indicates that three and one Trp residues are destroyed in air-and argon-saturated solutions, respectively. Polymers are devoid of all three activities of the Fpg protein, whereas the unpolymerized protein retains full activities. Matrix-assisted laser desorption/ionization experiments demonstrate that polymer formation is accompanied by the formation of short polypeptides containing the first 32 or 33 residues of the N-terminal domain. Theses polypeptides are most probably formed by the photolytic cleavage of Fpg protein induced by light absorption by the adjacent Trp-34 residue.