Purification and characterization of Escherichia coli formamidopyrimidine-DNA glycosylase that excises damaged 7-methylguanine from deoxyribonucleic acid

Abstract

A DNA glycosylase that excises 7-methylguanines with alkali-opened imidazole rings (formamidopyrimidines) from DNA has been purified more than 8000-fold from Escherichia coli cell extracts. The enzyme does not cleave 3-methyladenine, uracil, and intact 7-methylguanine from DNA. In assays containing pyrimidine analogues like oxauracil, 2,4,6-triaminopyrimidine, 2,5,6-triamino-2-hydroxypyrimidine sulfate, formamidopyrimidine, and 5-nitroso-2,4,6-triaminopyrimidine, only the two compounds showed end product inhibition of the enzyme. The enzyme has been named formamidopyrimidine-DNA glycosylase. It has a molecular weight of 30 000 and a Stokes radius of 26.4 A. The enzyme prefers double-stranded to single-stranded DNA and is stimulated by the presence of 0.1 M KCl in the reaction mixture.

8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.

“…An enzyme activity that cleaves 3' and 5' to this modified base, producing a single nucleotide gap in the modified strand, has recently been isolated from Escherichia coli (3). The general properties of this enzyme, designated 8-hydroxyguanine endonuclease, proved similar to those reported for the FPG protein of E. coli (4)(5)(6). The latter, also known as formamidopyrimidine (Fapy) DNA glycosylase, catalyzes release of imidazole ring-opened forms of guanine and adenine from alkylated or irradiated polynucleotides and DNA (4)(5)(6)(7)(8).…”

“…Purines undergo opening of the imidazole ring to create Fapy residues (18) or are hydroxylated at the C-8 position to form 8-oxodG and 8-oxodA (1,19). Imidazole ring-opening is facilitated by alkylation at N-7, followed by treatment with alkali, resulting in DNA containing Me-Fapy (5,8,20).…”

“…The base released during the reaction with 8-oxodG DNA was identified as 8-oxo-7,8-dihydroguanine . In view of the widespread distribution of 8-oxodG in cellular DNA (1), the demonstrated mutagenic properties of this lesion (9,10), and the identification of a bacterial gene coding for FPG protein (11), we propose that 8-oxodG represents a physiological substrate for this constituent DNA glycosylase of bacteria (4)(5)(6) and mammalian (12) cells.…”

Tumor progression to the metastatic state involves structural modifications in DNA markedly different from those associated with primary tumor formation

“…In a recent study (5), the transformation of primary breast tumors to the metastatic state was shown to involve a Ͼ2-fold increase in ⅐OH damage in DNA, as indicated by modified nucleotide base models comprising mutagenic 8-hydroxyadenine (6) and the putatively nonmutagenic ring-opened product 4,6-diamino-5-formamidopyrimidine (fapyadenine; refs. [7][8][9][10][11]. In addition, plots of the modified nucleotide base model log 10 (fapyadenine͞8-hydroxyadenine) versus the size of metastatic and nonmetastatic breast tumors revealed that the metastatic tumor DNA had significantly greater structural diversity than the nonmetastatic tumor DNA (P ϭ 0.01; ref.…”

DNA Repair Enzymes

“…It is eliminated from DNA by several mechanisms: spontaneous release by hydrolysis of the glycosylic bond, removal by TagH type enzymes, and opening of the im idazole ring (alkali-catalyzed) to produce 2,6-diamino-4-hydroxy-5N methylformamidopyrimidine (FAPY). An enzyme has been purified from E. coli (6,88) that removes FAPY as well as the adenine y-radiation product, 4,6-diamino-5-formamidopyrimidine (89) by a glycosylase action. This en zyme of Mr = 30,000 is specific for formamidopyrimidine in dsDNA.…”