Recognition of formamidopyrimidine by Escherichia coli and mammalian thymine glycol glycosylases. Distinctive paired base effects and biological and mechanistic implications.
The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates. 2, 6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide. The substrates containing Fapy:N pairs (N = A, G, C, T) as well as a Tg:A pair, a physiological substrate of Endo III, Endo VIII, and mNth1, were treated by the enzymes and nicked products were quantified by gel electrophoresis. The activity of Endo III and Endo VIII for Fapy varied markedly depending on the paired base, being the highest with G (activity relative to Tg = 0. 55 (Endo III) and 0.41 (Endo VIII)) and the lowest with C (0.05 (Endo III) and 0.06 (Endo VIII)). In contrast, mNth1 recognized all Fapy pairs equally well and the activity was comparable to Tg. The results obtained in the nicking assay were further substantiated by the analysis of the Schiff base intermediate using NaBH(4) trapping assays. These results indicate that Escherichia coli and mammalian Tg glycosylases have a potential activity to recognize Fapy. However, as demonstrated for Fapy:C pairs, their distinctive activities implicate unequal participation in the repair of Fapy lesions in cells.
Asagoshi
K
K
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Yamada
T
T
Okada
Y
Y
Terato
H
H
Ohyama
Y
Y
Seki
S
S
Ide
H
H
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008322:Mammals; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; C037301:N(5)-methyl-N(5)-formyl-2,5,6-triamino-4-hydroxypyrimidine; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli