Unraveling Mechanisms of Hydroxyurea-Induced Genotoxicity in C. elegans: The Interplay Between RNR Inhibition and Oxidative Stress

Abstract

Maintaining genome stability during DNA replication is essential for proper cell proliferation and organismal development. Disruption of nucleotide homeostasis and replication dynamics is a major source of replication stress, which can lead to genomic instability and cell death. Although the RNR inhibitor Hydroxyurea is widely used to induce replication stress, the relative contributions of nucleotide depletion and oxidative stress to its cellular effects remain incompletely understood. Maintaining intracellular dNTP pools is one of RNR's primary roles. Replication fork stalling and the activation of DNA damage response pathways result from HU's inhibition of RNR, which lowers dNTP availability. The molecular mechanisms driving HU-induced cellular stress were examined in this work using the model organism Caenorhabditis elegans, with a particular emphasis on the interaction between replication stress and reactive oxygen species buildup. Through a combination of HU treatments and RNA interference–mediated knockdown of RNR subunits, we demonstrate that RNR inhibition impairs DNA replication, as evidenced by reduced EdU incorporation in the germline. Replication stress was further confirmed by the accumulation of RPA-1 foci and by the activation of checkpoint signaling pathways, including CHK-1 and CDK-1 phosphorylation and ATM/ATR activation. These effects were accompanied by transcriptional activation of CEP-1/p53-dependent apoptotic regulators, including egl-1 and ced-13, ultimately resulting in increased germline apoptosis. In parallel, a dose-dependent increase in ROS levels upon HU treatment was observed, suggesting that oxidative stress represents an additional component of HU-induced cellular toxicity. Co-treatment with the antioxidant N-acetyl-L-cysteine (NAC) reduced ROS levels and partially alleviated apoptotic phenotypes, particularly at higher HU concentrations. Notably, RNAi-mediated depletion of RNR subunits recapitulated the key phenotypes observed following HU exposure. Collectively, these findings demonstrate a molecular connection between oxidative stress, DNA damage signalling, replication stress, and RNR suppression in vivo. These results provide new insight into the intricate relationship between replication dynamics and cellular stress responses and offer a deeper look into how nucleotide metabolic abnormalities lead to genomic instability.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences