Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase.

Mark C Johnson,Philip Zegerman,Geylani Can,Diana Alexander,Miguel Monteiro Santos

doi:10.7554/elife.63589

Mark C Johnson, Philip Zegerman + Show 3 more

Open Access

https://doi.org/10.7554/elife.63589

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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.

Highlights

It is vitally important that in every cell division the entire genome is replicated once and only once
Licensing is restricted to late mitosis/early G1 phase by the activity of the APC/C, which eliminates licensing inhibitors such as cyclin-dependent kinase (CDK) and geminin in this window of the cell cycle
Since the DNA damage checkpoint response can be activated in all phases of the cell cycle, we addressed whether the replication factors Sld3 and Dbf4 could be targeted by Rad53 outside of S-phase in vivo in budding yeast

Summary

Introduction

It is vitally important that in every cell division the entire genome is replicated once and only once. We show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-initiation of replication in G2/M phase and prevents premature firing of all origins, not just late origins, at the G1/S transition. Since the DNA damage checkpoint response can be activated in all phases of the cell cycle, we addressed whether the replication factors Sld3 and Dbf4 could be targeted by Rad53 outside of S-phase in vivo in budding yeast.

Results

Conclusion