Oxidative damage to DNA, reflected in the formation of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), may be important in mutagenesis, carcinogenesis and the ageing process. Kuchino et al. studied DNA synthesis on oligodeoxynucleotide templates containing 8-oxodG, concluding that the modified base lacked base pairing specificity and directed misreading of pyrimidine residues neighbouring the lesion. Here we report different results, using an approach in which the several products of a DNA polymerase reaction can be measured. In contrast to the earlier report, we find that dCMP and dAMP are incorporated selectively opposite 8-oxodG with transient inhibition of chain extension occurring 3' to the modified base. The potentially mutagenic insertion of dAMP is targeted exclusively to the site of the lesion. The ratio of dCMP to dAMP incorporated varies, depending on the DNA polymerase involved. Chain extension from the dA.8-oxodG pair was efficiently catalysed by all polymerases tested.

The major apurinic/apyrimidinic (AP) endonuclease of human cells, the Ape protein, incises DNA adjacent to abasic sites to initiate DNA repair and counteract the cytotoxic and mutagenic effects of AP sites. Here we address the determinants of Ape AP endonuclease activity using duplex DNA substrates that contain synthetic analogs of AP sites: tetrahydrofuranyl (F), propanediol (P), ethanediol (E), or 2-(aminobutyl)-1,3-propanediol (Q). The last of these, a branched abasic structure, was a poor substrate for which Ape had kcat > 1000-fold lower than for F. In contrast, the specificity constant (kcat/Km) for E or P of Ape purified from HeLa cells was only 5-8-fold lower than for F. Positioning a phosphorothioate ester immediately 5' to F inhibited Ape incision activity 20-fold (Rp isomer) or > 10,000-fold (Sp isomer). Although Ape did not have detectable endonuclease activity toward single-stranded substrates or unmodified double-stranded DNA, the enzyme displayed a low level of 3'-exonuclease activity for duplex DNA (< 0.03% of its AP endonuclease activity), which was influenced by the reaction conditions. The base positioned opposite F did not dramatically affect the cleavage efficiency of Ape, but an F:F arrangement was cleaved at approximately one-third of the efficiency of F:C. A 3'-mismatch diminished P and E cleavage only slightly and F not at all. A 5'-mismatch reduced the Ape cleavage rate 4-10-fold for F and approximately 100-fold for P and E. A series of substrates with F at different positions along the oligonucleotide showed that Ape requires > or = 4 base pairs 5' to the abasic site and > or = 3 base pairs on the 3'-side. The implications of these results for substrate recognition by Ape are discussed.

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase ␣. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase ␣ in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.Abasic sites in DNA arise spontaneously by hydrolysis, a process that can be accelerated by modification of purine bases (1) and by the catalytic action of N-glycosylases that remove damaged bases from DNA (2-4). The natural abasic site (Ab) 1 exists as an equilibrium mixture of the cyclic hemiacetal and open chain aldehyde forms of 2Ј-deoxyribose (see Fig. 1) and is subject to ␤-elimination. This chemical reaction leads to strand scission (5); for that reason, structural analogs of deoxyribose have often been used to explore biological properties of abasic sites in DNA (6 -10). Abasic site analogs include deoxyribitol, a model for the open chain form of the sugar (6, 7), and tetrahydrofuran, an isosteric and isoelectronic analog of deoxyribose (8, 9) that is cleaved by type II AP endonucleases, but is not subject to ␤-elimination (9).The miscoding properties of natural and synthetic abasic sites have been investigated under a variety of experimental conditions using randomly or site-specifically modified DNA (6,7,(11)(12)(13)(14)(15). The relative frequency of base incorporation opposite abasic sites and of chain extension from the 3Ј-primer terminus has also been reported (8, 9). While natural and synthetic abasic sites are structurally similar, their mutagenic potential has not been compared under the same experimental conditions.In Escherichia coli, synthesis past abasic sites in vitro (6 -9, 11-13) and in vivo (14, 15) is accompanied by preferential incorporation of dAMP opposite the lesion, a phenomenon known as the "A rule" (16). In eukaryotes, the presence of abasic sites in DNA is associated with differ...