Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1.
DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.
Speina
Elzbieta
E
Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Cieśla
Jarosław M
JM
Graziewicz
Maria-Anna
MA
Laval
Jacques
J
Kazimierczuk
Zygmunt
Z
Tudek
Barbara
B
D002243:Carbolines / Q000494:pharmacology*; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000494:pharmacology*; D014970:Xanthines / Q000494:pharmacology*
D002243:Carbolines; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011743:Pyrimidines; D014970:Xanthines; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C054700:6-thioxanthine; C019133:3-amino-1-methyl-5H-pyrido(4,3-b)indole; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli