PMID:1689309
| Citation |
Boiteux, S, O'Connor, TR, Lederer, F, Gouyette, A and Laval, J (1990) Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites. J. Biol. Chem. 265:3916-22 |
|---|---|
| Abstract |
The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli. Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites. To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid. The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside. From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties. In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5. When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion. Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule. The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E. coli. The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E. coli. |
| Links | |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Base Sequence; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Deoxyribonuclease IV (Phage T4-Induced); Endodeoxyribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Genes, Bacterial; Kinetics; Molecular Sequence Data; Molecular Weight; N-Glycosyl Hydrolases/genetics; N-Glycosyl Hydrolases/isolation & purification; N-Glycosyl Hydrolases/metabolism; Plasmids; Promoter Regions, Genetic; Protein Conformation; Substrate Specificity |
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