Revised Mechanism of Hydroxyurea Induced Cell Cycle Arrest and an Improved Alternative

Abstract

Replication stress describes various types of endogenous and exogenous challenges to DNA replication in S-phase. Stress during this critical process results in helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response is hydroxyurea (HU), a chemotherapeutic agent. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of dNTP synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, mediated by the replisome associated effector kinase Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast nuclei. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol , Pol {delta}, and Pol {varepsilon}, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron sulfur Fe-S clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.