Replication across Regioisomeric
Ethylated Thymidine
Lesions by Purified DNA Polymerases
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Abstract
Causal
links exist between smoking cigarettes and cancer development.
Some genotoxic agents in cigarette smoke are capable of alkylating
nucleobases in DNA, and higher levels of ethylated DNA lesions were
observed in smokers than in nonsmokers. In this study, we examined
comprehensively how the regioisomeric <i>O</i><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-ethylthymidine
(<i>O</i><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-EtdT, respectively) perturb DNA replication
mediated by purified human DNA polymerases (hPols) η, κ,
and ι, yeast DNA polymerase ζ (yPol ζ), and the
exonuclease-free Klenow fragment (Kf<sup>–</sup>) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that
hPol η and Kf<sup>–</sup> could bypass all three lesions
and generate full-length replication products, whereas hPol ι
stalled after inserting a single nucleotide opposite the lesions.
Bypass conducted by hPol κ and yPol ζ differed markedly
among the three lesions. Consistent with its known ability to efficiently
bypass the minor groove <i>N</i><sup>2</sup>-substituted
2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i><sup>2</sup>-EtdT, though it experienced great difficulty
in bypassing <i>N</i>3-EtdT and <i>O</i><sup>4</sup>-EtdT. yPol ζ was only modestly blocked by <i>O</i><sup>4</sup>-EtdT, but the polymerase was strongly hindered by <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT. LC–MS/MS
analysis of the replication products revealed that DNA synthesis opposite <i>O</i><sup>4</sup>-EtdT was highly error-prone, with dGMP being
preferentially inserted, while the presence of <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT in template DNA directed
substantial frequencies of misincorporation of dGMP and, for hPol
ι and Kf<sup>–</sup>, dTMP. Thus, our results suggested
that <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT
may also contribute to the AT → TA and AT → GC mutations
observed in cells and tissues of animals exposed to ethylating agents