Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions

摘要

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O²-alkylated thymidine (O²-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O²-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O²-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O²-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T→A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O²-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O²-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells.