Checkpoint inhibition of origin firing prevents DNA topological stress.

Esther C Morafraile,Mark C Johnson,George Allen,Christine Hänni,Nicola E Minchell,Philip Zegerman,Dave Lydall,Jonathan Baxter,Theresa Zeisner,Caroline Clarke,Lauren Carroll,Miguel M Santos,Peter Banks

doi:10.1101/gad.328682.119

Abstract

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

Highlights

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response
We generated alleles of SLD3 and DBF4 in budding yeast that cannot be phosphorylated by the checkpoint kinase Rad53 (Zegerman and Diffley 2010)
These alleles contain serine/threonine to alanine mutations at 38 sites in Sld3 and four sites in Dbf4 and are hereafter referred to as sld3-A and dbf4-A. These alleles are effective separation of function mutants because they are fully competent for their essential functions in replication initiation, yet they prevent checkpoint inhibition of origin firing while checkpoint activation and other functions of this pathway remain unaffected (Fig. 1B; Supplemental Fig. S1A; Zegerman and Diffley 2010)

Summary

Introduction

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. The first step in replication (prereplicative complex assembly or “licensing”) involves the loading of inactive double hexamers of the Mcm helicase at origins in G1 phase Initiation at these origins can only occur in S-phase due to the activation of the S-phase CDK (S-CDK) and Dbf4-dependent (DDK) kinases. Replication stress, for example, caused by DNA lesions, conflicts between DNA and RNA polymerase, or low levels of deoxynucleotide triphosphates (dNTPs), is an early event during tumorigenesis (Kotsantis et al 2018) Such stress leads to stalling of the replisome and activation of the checkpoint kinase ATR/Mec, which causes the subsequent activation of the effector kinase Chk in humans or Rad in yeast (Giannattasio and Branzei 2017). GENES & DEVELOPMENT 33:1539–1554 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org response to replication stress is called the S-phase, intraS-phase, or DNA replication checkpoint (Pardo et al 2017)

Methods

Results

Conclusion