DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase.
The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.
Castaing
B
B
Centre de Génétique Moléculaire, Université Pierre et Marie Curie, Gif/Yvette, France.
Boiteux
S
S
Zelwer
C
C
D001080:Apurinic Acid / Q000378:metabolism*; D001483:Base Sequence; D001894:Borohydrides; D043382:DNA-Formamidopyrimidine Glycosylase; D004586:Electrophoresis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction
D001894:Borohydrides; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D001080:Apurinic Acid; C025364:sodium borohydride; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli