Ultraviolet-B-induced damage to Escherichia coli Fpg protein.
We investigated the effect of UVB light (290 < or = lambda < or = 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 polyacrylamide 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.
Sidorkina
O M
OM
Groupe Réparation des Lésions Radio et Chimio-induites, Institut Gustave Roussy, Villejuif, France.
Kuznetsov
S V
SV
Blais
J C
JC
Bazin
M
M
Laval
J
J
Santus
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000528:radiation effects; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000528:radiation effects*; D010777:Photochemistry; D010782:Photolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli