Cdk1‐interacting protein Cip1 is regulated by the S phase checkpoint in response to genotoxic stress

Abstract

In eukaryotic cells a surveillance mechanism, the S phase checkpoint, detects and responds to insults that challenge chromosomal replication, arresting cell cycle progression and triggering appropriate events to prevent genomic instability. In the budding yeast Saccharomyces cerevisiae, Mec1/ATM/ATR, and its downstream kinase Rad53/Chk2, mediate the response to genotoxic stress. In this study, we place Cip1, a recently identified Cdk1 inhibitor (CKI), under the regulation of Mec1 and Rad53 in response to genotoxic stress. Cip1 accumulates dramatically in a Mec1 and Rad53 dependent manner upon replication stress. This increase requires the activity of MBF, but not the transcriptional activator kinase Dun1. At the protein level, stabilization of replication stress induced Cip1 requires continued de novo protein synthesis. In addition, Cip1 is phosphorylated at an S/TQ motif in a Mec1 dependent manner. Deletion of Cip1 affects proliferation in hydroxyurea containing plates. Significantly, the sensitivity is increased when the dosage of the G1 cyclin CLN2 is increased, compatible to a role of Cip1 as a G1-CDK inhibitor. In all, our results place Cip1 under the S phase checkpoint response to genotoxic stress. Furthermore, Cip1 plays a significant role to preserve viability in response to insults that threaten chromosome replication.

Dual Repressive Function by Cip1, a Budding Yeast Analog of p21, in Cell-Cycle START Regulation

“…The cDNA library was used as a template in real-time PCR using the Takara SYBR Premix Ex-Taq (Tli RNase H Plus) kit (Takara, RR420A). The primers for CLN2 and ACT1 were the same as in our previous study ( Zhang Z. et al, 2017 ).…”

“…This strategy for regulating CDK activity is conserved from yeast to human, but previously no p21 or p53 homolog had been identified in yeast. Recently, the Saccharomyces cerevisiae protein Cip1 was shown to regulate G1/S transition in both the unperturbed and stressed cell cycle ( Chang et al, 2017 ; Zhang Z. et al, 2017 ); accordingly, it was proposed as a functional analog of p21. Cip1 was originally identified as a G1-Cdk1 inhibitor that negatively regulates G1/S transition ( Ren et al, 2016 ).…”

Discovery of Pyruvate Kinase as a Novel Target of New Fungicide Candidate 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl-[1,2,4]-triazolo-[3,4-b][1,3,4]-thiadizole

“…The DNA-replication-checkpoint mutants Δmec1 and Δrad53 were obtained by courtesy of Professor Fan-Li Zeng (Hebei Agricultural University). 16 All the stains were cultured on YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 28 °C and 220 rpm.…”

The budding yeast Start repressor Whi7 differs in regulation from Whi5, emerging as a major cell cycle brake in response to stress

“…To overcome these stresses, cells process and integrate information from their environment into the cell cycle control network, in particular at Start ( Aldea et al, 2017 ; Ewald, 2018 ). In fact, multiple signalling pathways impinge on different regulators to promote or prevent passage through Start: molecular mechanisms connect the response to nutrient availability with Swi4 ( Amigoni et al, 2015 ), Whi5 ( Talarek et al, 2017 ), the cyclin-dependent kinase inhibitor Sic1 ( Moreno-Torres et al, 2015 ), the SBF-interacting proteins Msa1 and Msa2 ( Miles et al, 2016 ) or the cyclin Cln3 ( Gallego et al, 1997 ; Menoyo et al, 2013 ); osmotic stress targets Sic1 ( Escote et al, 2004 ), Whi5 ( Gonzalez-Novo et al, 2015 ) and Cip1 ( Chang et al, 2017 ); and genotoxic stress affects transcriptional factors Nrm1 ( Travesa et al, 2012 ), Swi6 ( Sidorova and Breeden, 1997 ) as well as Cip1 ( Zhang et al, 2017 ). Therefore, multiple regulatory pathways might cooperate in regulating the G1-S transition for cells to cope with environmental changes.…”