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Aging, The Molecular Concepts


Abasic sites in DNA arise spontaneously by a hydrolytic process, which is markedly accelerated by chemical modification of DNA bases. Approximately 100 000 abasic sites per cell are formed each day (4). Damaged DNA bases are removed by DNA N-glycosylases, generating abasic sites as an intermediate during base excision repair. These lesions may play an important role in spontaneous and chemical mutagenesis (5). During DNA synthesis, dAMP is inserted preferentially opposite abasic sites in DNA. This phenomenon, known as the A-rule (27), is associated with replication past non-instructive lesions in vitro (28-33) and in vivo (34, 35). Synthetic abasic sites lacking the deoxyribose moiety (propanyl, ethanyl) direct the preferential incorporation of dAMP opposite the lesion (31), indicating that hydrogen bonding involving the lesion does not play a major role in the selection of dAMP. Preferential insertion of dAMP opposite abasic sites appears to be an intrinsic property of all DNA polymerases, possibly including DNA polymerase ζ of S. cerevisiae (36). The natural abasic site in DNA consists of a 2'-deoxyribose moiety linked to neighboring bases through 3' and 5' phosphodiester bonds. In aqueous solution, this structure is represented by an equilibrium mixture of tautomers consisting primarily of α and β anomers of deoxyribofuranose (dR) and accompanied by a low level of the partially hydrated (open chain) aldehydo form. Structures of this type are subject to β-elimination, leading to strand scission. The synthetic abasic sites shown in Fig. 4.2 are structural analogs of the natural abasic site. The tetrahydrofuran moiety (F) is an analog of the predominant (cyclic) form of the natural abasic site. The propanyl moiety (P) represents an anucleosidic site with a three-carbon phosphodiester backbone. Propanyl and ethanyl (E) analogs (Fig. 4.2) are models for the minor (acyclic) form of the abasic sites. Embedded in duplex DNA, synthetic abasic sites are substrates for bacterial and mammalian AP endonucleases (31, 37). In single strand DNA templates, these model lesions have been used to study miscoding properties of abasic sites (30, 31). In both systems, dAMP is preferentially inserted opposite natural and synthetic abasic sites. Kunz et al. (38) observed an increase of G—>C transversions in AP endonuclease deficient strains of S. cerevisiae. The mutagenic potential of abasic sites also has been demonstrated by introducing a single strand shuttle plasmid vector containing a single abasic site into E. coli or mammalian cells. Using this system, Sarasin and co-workers reported that the "A rule" is not observed in simian kidney (COS) cells (39). Using a duplex vector, Takeshita & Eisenberg (40) found that dAMP is preferentially incorporated opposite synthetic abasic sites in mammalian cells. Lawrence et al. (41) report that the preferred nucleotide incorporated in yeast is dCMP while dAMP is inserted opposite abasic sites in E. coli (35). Further studies are needed to explain the apparent deviations from the A-rule.

Under physiological conditions, abasic sites are rapidly generated and rapidly repaired; thus, isolated abasic sites are unlikely to contribute significantly to spontaneous or chemically induced mutagenesis in repair-competent cells. Abasic sites could play a more important role where complex DNA damage is involved. For example, bleomycin and neocarzinostatin cleave duplex DNA, generating bistrand lesions composed of an abasic site and a single strand gap positioned on opposite strands. Mutagenic properties of these radiomimetic agents are attributed, in part, to the abasic site moiety (42, 43). Synthetic abasic sites were used to explore mutagenesis at bistrand lesions in duplex DNA (40). While single abasic sites were rapidly repaired, abasic sites in a bistrand configuration were highly mutagenic.

Fig. 4.3.
Fig. 4.3. Mechanism for frameshift deletion mutation (46).

Two molecular events appear to be involved; repair of an abasic site during translesional synthesis and concomitant incorporation of dAMP opposite the other lesion. Abasic sites also are associated with frameshift deletion mutations (44). An in vitro system was used to quantify deletions and base substitutions formed during DNA synthesis (45). The marked influence of sequence context on frameshift mutagenesis was demonstrated by modifying the bases flanking the lesion (46). The frequency of nucleotide insertion opposite natural and synthetic abasic sites and of chain extension from the 3' primer terminus was established by steady state kinetics. The ability of an abasic site to generate one-base and two-base frameshift deletions was determined primarily by the nature of the base inserted opposite the abasic site with respect to the sequence context in which the lesion is embedded and the overall rate of translesional DNA synthesis. Misinsertion of bases opposite the lesion precedes misalignment of the primer-template. Anucleosidic sites generate more deletions than cyclic analogs of deoxyribose. These findings are in accord with the general model we have proposed for frameshift deletion mutagenesis (Fig. 4.3), building on earlier studies by Kunkel & Soni (47) and Boosalis et al. (48).

Fig. 4.4.
Fig. 4.4. Structures of exocyclic DNA adducts

In summary, single abasic sites in duplex DNA are rapidly repaired in cells; in a bistrand configuration, as exists in clustered lesions produced by ionizing radiation (49) and radiomimetic agents, abasic sites are highly mutagenic. Abasic sites block progression of DNA polymerase during DNA synthesis; however, some translesional synthesis is observed with dAMP preferentially incorporated opposite the lesion. Base substitution mutations may occur when a base is inserted opposite an abasic site; however, depending on the base 5' to the lesion and the degree of block, frameshift deletions may be generated by the mechanism shown in Fig. 4.3.

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