Revised mechanism of hydroxyurea- induced cell cycle arrest and an improved alternative

• 成果类型: Article
• 署名作者: Shaw, Alisa E.; Mihelich, Mattias N.; Whitted, Jackson E.; Reitman, Hannah J.; Timmerman, Adam J.; Tehseen, Muhammad; Hamdan, Samir M.; Schauer, Grant D.
• 署名单位: Colorado State University System; Colorado State University Fort Collins; King Abdullah University of Science & Technology
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13895
• DOI: 10.1073/pnas.2404470121
• 发表日期: 2024-10-15
• 关键词: dna-replication stress; ribonucleoside-diphosphate reductase; iron-sulfur cluster; c-terminal domain; s-phase; chromosome integrity; triphosphate pools; crystal-structure; escherichia-coli; inhibition

摘要

Replication stress describes endogenous and exogenous challenges to DNA replication in the S- phase. Stress during this critical process causes 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 in yeast is hydroxyurea (HU), a clinically used chemotherapeutic. The primary mechanism of S- phase checkpoint activation by HU has thus far been considered to be a reduction of deoxynucleotide triphosphate synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, facilitated by the replisome- associated mediator 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 and human cells and show that antioxidants restore growth of yeast in HU. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol alpha, Pol delta, and Pol 8, 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.