Replicative Bypass of O2-alkylthymidines Lesions In Vitro

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, O 2 -alkylthymidines ( O 2 -alkyldT) are known to be persistent and poorly repaired in mammalian systems, and have been shown to accumulate in 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 O 2 -alkyldT lesions, with the alkyl group being a Me, Et, n Pr, i Pr, n Bu, i Bu or s Bu, 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 O 2 -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 O 2 -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 O 2 - i PrdT > O 2 - i BudT > O 2 - s BudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O 2 -alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.