The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5¢-dangling modi®ed nucle-otide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage-speci®c endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2¢-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2¢-deoxyuridine, alpha-2¢-deoxyadenosine and alpha-thymidine adducts, generating 3¢-hydroxyl and 5¢-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly ef®cient. The substrate speci®city and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed