Incorporation of dA opposite N3-ethylthymidine terminates in vitro DNA synthesis

Abstract

N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine. The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phosphite triester method. The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end. The template DNA, which corresponds to a specific sequence at gene G of bacteriophage phi X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 microM dNTP and 5 mM Mg2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96% immediately 3' to N3-Et-dT and 4% after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product). DNA replication past the lesion (postlesion synthesis) was negligible. Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35% at 200 microM. Postlesion synthesis remained negligible. DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the "A" rule in mutagenesis. Formation of the N3-Et-dT.dA base pair at the 3'-end of the growing chain terminated DNA synthesis. These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents.