Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil
incorporation contributing to their antiproliferative effect. Still, it is not yet
characterized if uracil incorporations have any positional preference. Here, we aimed
to uncover genome-wide alterations in uracil pattern upon drug treatments in human
cancer cell line models derived from HCT116. We developed a straightforward U-DNA
sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging.
Using a novel robust analysis pipeline, we found broad regions with elevated probability
of uracil occurrence both in treated and non-treated cells. Correlation with chromatin
markers and other genomic features shows that non-treated cells possess uracil in
the late replicating constitutive heterochromatic regions, while drug treatment induced
a shift of incorporated uracil towards segments that are normally more active/functional.
Data were corroborated by colocalization studies via dSTORM microscopy. This approach
can be applied to study the dynamic spatio-temporal nature of genomic uracil.
