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Aging, The Molecular Concepts


1,N6-ethenodeoxyadenosine (εdA), 3,N4-ethenodeoxycytidine (εdC) and 1,N2- and N2, 3-ethenodeoxyguanosine (εdG) adducts found in human and rat liver DNA (Fig. 4.4) are formed from endogenous metabolic processes such as lipid peroxidation (50, 51). N2-(l,3-propanyl)deoxyguanosine (PdG) is an analog for the several exocyclic adducts produced when α, β-unsaturated aldehydes such as acrolein react with DNA (52).

The miscoding properties and mutagenic potential of exocyclic DNA adducts in E. coli and COS7 cells (53-55) was explored. In E. coli, εdC was weakly mutagenic while εdA had a mutation frequency of <0.7% (Table 4.II). When host cells were UV-irradiated prior to transformation, the mutation frequency for εdC increased while that of εdA was unaffected. The predominant mutations observed were εdC—>T and εdC—>A. In E. coli, PdG directed the incorporation of dAMP, resulting in 100% PdG—>T transversions. In UV-irradiated cells, this exclusive specificity diminished and PdG—>A transitions also were observed. The 3'—>5' exonuclease activity associated with the ε subunit of DNA polymerase III catalyzes removal of bases misincorporated during DNA replication.

Table 4.II
Mutagenic specificity of exocyclic DNA adducts1
DNA adductHostTargeted events (%)DNA adduct—>C, A, T or GFrequency of targeted mutations (%)
1 Adducts were incorporated into single-stranded DNA and replicated in host cells. Targeted events were analyzed. See Moriya et al. (53) for detailed procedure.

When E. coli NR9232, a mutant strain carrying a nonfunctional ε subunit, was used as a host cell, the frequency of targeted mutations for εdC was increased to 33% (53). This value in non-irradiated cells is equal to that observed for εdC following UV-irradiation of wild type AB1157. The increased mutation frequency reflected mainly εdC—>A transversions; the marked effect of the ε subunit on mutagenesis indicates that translesional synthesis is catalyzed by DNA polymerase III. These results suggest that dT, as well as non-mutagenic dG, is incorporated opposite εdC with dT removed preferentially by the 3'—>5' exonuclease activity of the ε subunit in wild type cells. In contrast, mutation frequency did not increase with or without UV irradiation when plasmids containing εdA were introduced into NR9232, indicating that dT is inserted exclusively opposite this adduct.

When the same constructs were transfected into simian kidney (COS7) cells, frequencies of targeted mutations for the two etheno adducts were high: 70% for εdA and 81% for εdC (53, 54). εdA—>G transitions predominated, followed by εdA—>A (non-mutagenic), εdA—>T, and εdA—>C. εdC—>A transversions were most commonly observed, followed by εdC—>T, εdC—>C (non-mutagenic), and εdC—>G. Thus, preferences for bases inserted opposite exocyclic adducts in COS cells are C>T>A>G for εdA and T>A>G>C for εdC. In spite of the powerful miscoding properties demonstrated in E. coli, PdG was not highly mutagenic in COS cells (Table 4.II). Targeted events included PdG—>G, PdG—>T, and PdG—>C. PdG—>A transitions were not observed; thus, the frequency of bases incorporated opposite PdG was C>A>G. Structural studies reveal that the nonmutagenic εdA(anti):T(anti) pair forms a nonplanar alignment without hydrogen bonds. In contrast, εdA(syn):dG(anti), generating in the rare εdA—>C transversions, is stabilized by two hydrogen bonds (56). The structure of εdA:dC, the major event in COS cells, remains to be determined. εdC in either syn or anti conformation is incapable of forming more than one hydrogen bond in B-DNA (57, 58); nevertheless, dG, dT, and dA are readily incorporated opposite εdC during DNA synthesis. Remarkably, the fidelity of DNA synthesis in E. coli remains high. At physiological pH, approximately half the population of PdG molecules adopt a syn conformation, forming a PdG(syn):dA(anti) pair with two hydrogen bonds (59). The stability of this structure accounts for the exclusive PdG—>T transversions observed in non-irradiated E. coli even though PdG is a strong block to DNA synthesis. PdG:dC is the dominant pairing event in COS cells.

In summary, εdC and εdA are much more mutagenic in COS cells than in E. coli, while PdG has an opposite effect, suggesting that miscoding by these exocyclic adducts depends on host factor(s). Mutational specificity is determined by a complex interplay between DNA polymerase, adduct structure, and the DNA sequence context. DNA polymerase III is responsible for the mutagenic events observed in E. coli. In mammalian (COS7) cells, it is not clear which DNA polymerase is involved. In E. coli, significant fidelity is retained even with exocyclic adducts that do not form hydrogen bonds.

The most common forms of endogenous DNA damage, including 8-oxoguanine, abasic sites, and exocyclic DNA adducts, are mutagenic in mammalian cells, generating base substitutions and in some cases frameshift deletions. Such mutations may accumulate over time, contributing to the alterations in protein structure and function associated with the aging process.

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