AP site structural determinants for Fpg specific recognition

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) si te analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E. coli and L. lactis enzymes (K-Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study (K-Dapp > 2.8 nM), The 1,3-propanediol (Pr) residue-containing DNA s eems to be the minimal AP site structure allowing a Fpg specific DNA bindin g, since the ethyleneglycol residue is not specifically bound by these enzy mes. The newly described cyclopentanol residue is better recognized than te trahydrofuran (for the E.coli Fpg, K-Dapp = 2.9 and 25 nM, respectively), T hese results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4' -hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl ra dical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposit e the lesion on the undamaged complementary strand, This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.