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Duplex oligodeoxynucleotides with synthetic analogs of abasic sites were used to study the specificity of the abasic endonucleases of Escherichia coli. The apparent Km values of exonuclease III for the tetrahydrofuranyl, propanyl, and deoxyribosyl substrates varied only somewhat (20-140 nM) in either Mg2+ or Ca2+ and were similar to those for endonuclease IV. In Mg2+, exonuclease III had a turnover number 4-13-fold higher than measured for endonuclease IV (ranging 5.6-18 min-1), but was lowered in Ca2+ to values similar to those for endonuclease IV. The rate of cleavage of tetrahydrofuranyl (F) substrate by both enzymes was unaffected by the base in the opposite strand or its replacement by a tetrahydrofuranyl moiety. A C:C mismatch on the 5' but not the 3' side of F strongly inhibited cleavage by exonuclease III in Ca2+, while mismatches on both sides were required to diminish endonuclease IV cleavage significantly. A phosphorothioate ester linked 5' to the tetrahydrofuranyl moiety inhibited both enzymes, with the Rp stereoisomer most effective. Endonuclease IV bound stably to duplex substrates containing the Rp phosphorothioate in the presence of poly(dI-dC). Although the apurinic/apyrimidinic-cleaving activities of endonuclease IV and exonuclease III have some common features they also differ in their specific interactions with DNA containing abasic sites.
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