An essential role for dNTP homeostasis following CDK-induced replication stress.

Chen-Chun Pai,Jürg Bähler,Stephen E Kearsey,Timothy C Humphrey,Kuo-Feng Hsu,Christopher J Schofield,Rachel Deegan,Sarah E Wilkins,Antony M Carr,Charalampos Rallis,Sophia X Pfister,Nagore De Leόn,Lisa K Folkes,Samuel C Durley,Andrea Keszthelyi

doi:10.1242/jcs.226969

Abstract

ABSTRACTReplication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a ‘dNTP supply and demand’ model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

Highlights

Replication stress, in which DNA replication forks stall, is a source of genome instability and a common feature of cancer cells (Gaillard et al, 2015)
We demonstrate that mutations that cause deoxyribonucleoside triphosphate (dNTP) homeostasis defects, resulting from either loss of Set2 or the DNA integrity checkpoint inactivation, are synthetic lethal with CDKinduced replication stress resulting from Wee1 inactivation
We found that the replication stress associated with Wee1 inactivation alone, or in combination with set2Δ, resulted in DNA damage (Rad52–GFP foci formation)

Summary

Introduction

Replication stress, in which DNA replication forks stall, is a source of genome instability and a common feature of cancer cells (Gaillard et al, 2015). Received 29 October 2018; Accepted 2 January 2019 multiple events including physical blockage of replication fork progression, deregulation of the replication initiation or elongation complexes or through deoxyribonucleoside triphosphate (dNTP) depletion (Dobbelstein and Sorensen, 2015; Zeman and Cimprich, 2014). Cells respond to such events by triggering checkpointdependent responses to facilitate DNA replication restart (Mazouzi et al, 2014). Unresponsive stalled forks can be subject to endonucleolytic cleavage by Mus81–Eme, generating a DNA end, which is targeted for homologous recombination (HR) (Hanada et al, 2007; Roseaulin et al, 2008)

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Results

Conclusion