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


O(2)- and O(4)-methylthymidine (O(2)-MdT and O(4)-MdT) can be induced ...
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.




new topics/pols set partial results complete validated


No results available for this paper.

Entry validated by:

Using Polbase tables:


Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).


It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.