Replicative Bypass of O2-Alkylthymidine Lesions in Vitro

Abstract

DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, O²-alkylthymidines (O²-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific N-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of O²-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, were capable of bypassing all O²-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol η exhibited high frequencies of misincorporation of dCMP opposite those O²-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of O²-iPrdT > O²-iBudT > O²-sBudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O²-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Figure 1

(a) The structures of the O²-alkyldT lesions examined in this study and (b) the primer-template complex used for the in vitro primer extension and steady-state kinetic assays, X= dT, O²-MedT, O²-EtdT, O²-nPrdT, O²-iPrdT, O²-nBudT, O²-iBudT or O²-sBudT.

Figure 2

Representative gel images from the primer extension assays under standing-start conditions for primer-template complexes containing an unmodified dT, O²-MedT, O²-EtdT, O²-nPrdT, O²-iPrdT, O²-nBudT, O²-iBudT or O²-sBudT with human Pol η. The final concentration of the primer-template complex was 10 nM, and the final concentrations of human Pol η are indicated.

Figure 3

Representative gel images for steady-state kinetic assays in measuring the individual nucleotide incorporation opposite unmodified dT (a), O²-nPrdT (b) and O²-nBudT (50 nM) (c) with human Pol η. The final concentration of the primer-template complex was 10 nM, and the final concentration of human Pol η was 5 nM. The highest concentrations of individual dNTPs used are indicated in the figure, and the concentration ratio between neighboring lanes was 0.50.

Figure 4

The efficiencies for the human Pol η-catalyzed insertion of the correct nucleotide, dAMP, opposite unmodified dT, O²-MedT, O²-EtdT, O²-nPrdT, O²-iPrdT, O²-nBudT, O²-iBudT or O²-sBudT (relative to dT). The results represent the mean ± standard deviation of results from at least three independent measurements.

Figure 5

Relative efficiencies for the Pol η-mediated nucleotide incorporation opposite dT, O²-MedT, O²-EtdT, O²-nPrdT, O²-iPrdT, O²-nBudT, O²-iBudT and O²-sBudT.