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Homogeneous Escherichia Coli FPG Protein. A DNA Glycosylase Which Excises Imidazole Ring-Opened Purines and Nicks DNA at apurinic/apyrimidinic Sites

Homogeneous Escherichia Coli FPG Protein. A DNA Glycosylase Which Excises Imidazole Ring-Opened Purines and Nicks DNA at apurinic/apyrimidinic Sites

S Boiteux, T R O'Connor, F Lederer, A Gouyette, J Laval

J Biol Chem. 1990 Mar 5;265(7):3916-22.

PMID: 1689309

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP78783536	Fpg Protein from Escherichia coli		78783-53-6	Price

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