Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase
- Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, United Kingdom
Abstract
Checkpoints maintain the order of cell cycle events during DNA damage or incomplete replication. How the checkpoint response is tailored to different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in budding yeast occurs by Rad53-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed, we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M, preventing gene amplification. In addition, we show that inhibition of Sld3 and Dbf4 in G1 prevents premature initiation at all origins at the G1/S transition. This study redefines the scope of the 'S-phase checkpoint' with implications for understanding checkpoint function in cancers that lack cell cycle controls.
Data availability
Sequencing data has been deposited in GEO under the accession code GSE159122 and GSE163571
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Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phaseNCBI Gene Expression Omnibus, GSE159122.
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Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phaseNCBI Gene Expression Omnibus, GSE163571.
Article and author information
Author details
Miguel Monteiro Santos
Funding
Worlwide Cancer Research (AICR 10-0908)
- Mark C Johnson
- Geylani Can
- Miguel Monteiro Santos
- Diana Alexander
- Philip Zegerman
Wellcome Trust (107056/Z/15/Z)
- Mark C Johnson
- Geylani Can
- Miguel Monteiro Santos
- Diana Alexander
- Philip Zegerman
Biotechnology and Biological Sciences Research Council (BB/M011194/1)
- Miguel Monteiro Santos
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Tim Formosa, University of Utah School of Medicine, United States
Version history
- Received: September 29, 2020
- Accepted: January 4, 2021
- Accepted Manuscript published: January 5, 2021 (version 1)
- Version of Record published: January 13, 2021 (version 2)
Copyright
© 2021, Johnson et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase
eLife 10:e63589.
https://doi.org/10.7554/eLife.63589
Further reading
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We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs.
