Ultraviolet-B-induced damage to Escherichia coli Fpg protein

We investigated the effect of UVB light (290 less than or equal to lambda l ess than or equal to 320 nm) on the structure and enzymatic activities of E scherichia coli Fpg protein (2,6-diamino-4-hydroxy-5N-methylformamidopyrimi dine-DNA glycosylase), a DNA repair enzyme containing a zinc finger motif a nd five chromophoric Trp residues. Irradiation with UVB light of air-satura ted pH 7.4 buffered aqueous solutions of Fpg induces the formation of polym ers as shown by sodium dodecyl sulfate polyacrylamide gel electrophoretic a nalysis. In argon-saturated solutions, polymer formation produces a precipi tate. The polymerization quantum yield is 0.07 +/- 0.01 and 0.15 +/- 0.02 i n 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, resp ectively, Polymers are devoid of all three activities of the Fpg protein, w hereas the unpolymerized protein retains full activities. Matrix-assisted l aser desorption/ionization experiments demonstrate that polymer formation i s accompanied by the formation of short polypeptides containing the first 3 2 or 33 residues of the N-terminal domain. Theses polypeptides are most pro bably formed by the photolytic cleavage of Fpg protein induced by light abs orption by the adjacent Trp-34 residue.