Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichiacoli

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E.co li catalyzes excision of several damaged purine bases, including 8-oxoguani ne 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 ef ficiently, not only from double-stranded, but also from single-stranded oli godeoxynucleotides. The Michaelis constants (K-M) of a range of single-stra nded oligodeoxynucleotides (0.55-1.3 mu M) were shown to be 12-170 times hi gher that those for corresponding double-stranded oligodeoxynucleotides (K- M = 6-60 nM). Depending on the position of the 8-oxoguanine within the olig odeoxynucleotides, 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 effecti vely 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-irra diation in the presence of photoreactive analogs of single-stranded and dou ble-stranded oligodeoxynucleotides. Specific and nonspecific single-strande d and double-stranded oligodeoxynucleotides essentially completely prevente d the covalent binding of Fpg by the photoreactive analog. All these data a rgue for similar interactions occurring in the DNA binding, cleft of the en zyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides diffe r by no more than 2 orders of magnitude. Addition of the second complementa ry chain increases the affinity of the first single-stranded chain by a fac tor of similar to 10. It is concluded that Michaelis complex formation of F pg with DNA containing 8-oxoG cannot alone provide the major part of the en zyme specificity, which is found to lie in the k(cat) term for catalysis; t he reaction rate being increased by 6-7 orders of magnitude by the transiti on from nonspecific to specific oligodeoxynucleotides.