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Chem Res Toxicol. 2012 Nov 19;25(11):2523-31. doi: 10.1021/tx300325q. Epub 2012 Nov 8.

In-vitro replication studies on O(2)-methylthymidine and O(4)-methylthymidine.

Author information

1
Department of Chemistry, University of California, Riverside, CA 92521-0403, USA.

Abstract

O(2)- and O(4)-methylthymidine (O(2)-MdT and O(4)-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O(2)-MdT and O(4)-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf(-)), human DNA polymerase κ (pol κ), and Saccharomyces cerevisiae DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf(-) and pol η preferentially incorporated the correct nucleotide (dAMP) opposite O(2)-MdT, while O(4)-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite O(2)-MdT and O(4)-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if left unrepaired, O-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents.

PMID:
23113558
PMCID:
PMC3502631
DOI:
10.1021/tx300325q
[Indexed for MEDLINE]
Free PMC Article

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