Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/ apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg: Like its Escherichia coli homolog, endonuclease III (Nth), hNTH1 1 is a bifunctional DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase that removes ring-saturated pyrimidines, be they hydrated, reduced, or oxidized, from the DNA backbone as the initial step of base excision repair (BER) of such modified residues (1). The oxidation product of thymine, 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol (Tg)) is a widely studied model substrate for hNTH1 and catalyzes its release from DNA via its DNA N-glycosylase activity. This enzyme activity is mediated via formation of a transient Schiff base (imino) enzyme-DNA intermediate, an example of covalent catalysis and a characteristic of all known bifunctional DNA N-glycosylases/AP lyases (4, 5). The Schiff base moiety has been hypothesized to be required for the enzymatic catalysis of the -elimination reaction, which effects DNA strand cleavage 3Ј to the abasic (AP) site formed as a product of the release of the base from the 2Ј-deoxyribose moiety to which it was linked. The hydrolysis of the Schiff base intermediate can occur in the absence of or following enzyme-catalyzed -elimination (AP lyase activity), resulting in DNA strand cleavage. The enzymatic catalysis of -elimination by DNA N-glycosylases/AP lyases has been shown to be initiated via abstraction of the deoxyribose pro-S-2Ј-hydrogen by a basic amino acid in the enzyme active site. Several factors, including pH, can affect the efficiency of the AP lyase step by affecting substrate binding and proton abstraction (5).ABased on the results of studies with Nth, the two activities of hNTH1 (DNA N-glycosylase and -elimination catalysis) require the formation of the Schiff base intermediate and were thought to occur concomitantly. However, data from our laboratory (1) and from other laboratories (6 -8) indicate that DNA N-glycosylase and -elimination catalysis by mammalian members of the endonuclease III enzyme superfamily are not concurrent under the assay conditions employed. We were the first to report that the DNA N-glycosylase and AP lyase activities of hNTH1 are not concurrent, i.e. that the rate of AP lyase-mediated strand cleavage is much slower than the rate of DNA N-glycosylase-mediated base release. These results are similar to the non-concurrence of base release and strand cleavage reported for the mammalian 8-oxoguanine-DNA N-glycosylase homolog OGG1 (8). In this report, we present data demonstrating that the dissociation of the two activities of hNTH1 is dependent on the nature of the orphan base opposite the Tg residue in DNA.In our studies of the properties of hNTH1, we have focused