Structural Requirements of Double and Single Stranded DNA Substrates and Inhibitors, Including a Photoaffinity Label, of Fpg Protein From Escherichia Coli

Abstract

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E.coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E.coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (K_M) of a range of single-stranded oligodeoxynucleotides (0.55−1.3μM) were shown to be 12–170 times higher that those for corresponding double-stranded oligodeoxynucleotides (K_M = 6–60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of ∼ 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the k_cat , term for catalysis; the reaction rate being increased by 6–7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.