The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coil, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase ofE. coil has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimiidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. Thefpg gene is different from the xth, nth, and nfo genes of E. colt, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ionexchange, molecular weight exclusion, and hydrophobicity.The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved .8 elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coil DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coil exonuclease m. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'-and 3'-phosphomonoester nucleotides and the release of the deoxyribose.The major lesion formed in DNA treated with alkylating agents is N7-alkylguanine (1). N7-Methylguanine is apparently tolerated by the cell, since this base does not block in vitro synthesis (2-4) and persists in DNA over generations (5). This lesion, however, may undergo imidazole ring opening to yield a 2,6-diamino-4hydroxy-5N-formamidopyrimidine (Fapy) residue or destabilize the glycosylic bond to yield an apurinic/apyrimidinic (AP) site (6)(7)(8). In contrast to N7-methylguanine, Fapy residues block in vitro DNA synthesis one base before the lesion (2, 3), and thus by analogy to N3-methyladenine are potentially lethal (4, 9). Treatment of DNA with y-radiation also introduces breaks between the C8 and N9 of the imidazole ring of adenine and guanine (10).In DNA, Fapy lesions are repaired by the base excision pathway. The first step in this pathway is the cleavage of the glycosylic bond by a Fapy-DNA glycosylase to release a free Fapy base and generate an AP site. These sites are noninstructive, premutagenic lesions (11,12) (20), or at the C8 such as for N-hydroxy-2-aminofluorene (S. Boiteux, and J.L., unpublished results) and also adenines, whose imidazole rings are opened by y-radiation (10).We found that a purified fraction containing Fapy-DNA glycosylase activity also showed an activity that nicked DNA at AP ...