Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase

Li Phing Liew,Zun Yi Lim,Matan Cohen,Ziqing Kong,Lisette Marjavaara,Andrei Chabes,Stephen D Bell

doi:10.1016/j.celrep.2016.10.024

Li Phing Liew, Zun Yi Lim + Show 5 more

Open Access

https://doi.org/10.1016/j.celrep.2016.10.024

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Journal: Cell Reports	Publication Date: Nov 1, 2016
Citations: 23	License type: cc-by

Affiliation: Indiana University Bloomington, Umeå University

Abstract

SummaryIn many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

Highlights

Hydroxyurea (HU) is widely used as a reagent to promote replication fork stalling in a range of organisms (Krakoff et al, 1968; Young and Hodas, 1964)
Sulfolobus Growth Is Inhibited by HU Treatment Members of the hyperthermophilic archaeal genus Sulfolobus encode a gene, annotated as nrdB, encoding a protein homologous to the class I ribonucleotide reductase (RNR) small subunit
The sole Sulfolobus large-subunit RNR homolog (SSO0929) appears most closely related at the primary sequence level to class II and is conserved across the archaeal domain including many lineages that lack the R2-like proteins (Figure S1). It was unclear whether Sulfolobus RNR would be sensitive to treatment of cells with HU

Summary

Introduction

Hydroxyurea (HU) is widely used as a reagent to promote replication fork stalling in a range of organisms (Krakoff et al, 1968; Young and Hodas, 1964). Depletion of dNTP pools leads to replication fork stalling. This triggers induction of the MazF and RelE toxins that in turn lead to improper translation of proteins and consequent membrane stress. Upon superoxide conversion to hydrogen peroxide, the reaction of hydrogen peroxide with free ferrous iron leads to hydroxyl radical generation via the Fenton reaction. This effect is likely exacerbated by an influx of iron, triggered by a response to the requirement to synthesize increased levels of RNR (Davies et al, 2009). In addition to HU’s action via the class I RNRs, it has been revealed that HU and its breakdown products can have a range of additional effects on cells. Kuong and Kuzminov (2009) have revealed that HU breaks down in aqueous solution to form nitrous oxide, cyanide, and peroxides, leading to the proposal that these compounds may contribute to the toxicity of HU

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