Abstract 2065 A Revised Mechanism of Replication Stress by Hydroxyurea

Replication stress encompasses various types of endogenous and exogenous challenges to DNA replication during S-phase. Stress during this critical phase results in helicase-polymerase decoupling, which triggers the S-phase checkpoint response that orchestrates global replication fork stalling and cell cycle stalling. One widely used exogenous replication stressor employed to induce the checkpoint response is hydroxyurea (HU), an FDA-approved drug that has been shown to inhibit dNTP synthesis by inhibition of ribonucleotide reductase. Here we show an alternative mechanism for HU-induced replication stress in yeast cells, which likely accounts for the majority of its activity as a replication stress agent. We demonstrate HU generates copious nuclear Reactive Oxygen Species (ROS), which in turn strongly inhibit all three replicative polymerase complexes via their integral Fe-S cluster. The results have broad implications for interpreting data generated with HU. Finally, we offer a genetically encoded alternative to HU that is more readily reversible and does not result in ROS generation, allowing researchers to study replication stress with improved specificity towards replication in the absence of the confounding variable of global oxidative stress. This project was funded by the NIH (R35 GM147105)