Abstract RB1 (retinoblastoma) members control the G1/S commitment as transcriptional repressors in eukaryotic cells. Here we uncover that an extra copy of RB1 equivalent ( WHI7 or WHI5 ) is sufficient to bypass the indispensability of the central genomic checkpoint kinases Mec1 ATR -Rad53 CHK1 in Saccharomyces cerevisiae . Mec1-Rad53 directly phosphorylate Whi7/5, antagonizing their nuclear export or protein turnover upon replication stress. Through in vitro reconstitution, we show that Whi7 C-terminus directly binds and hinders S-CDK-Cks1 from processively phosphorylating Sic1. By microfluidic single-cell real-time quantitative imaging, we demonstrate that both Whi7 and Whi5 are required to flatten the degradation curve of the major S-CDK inhibitor Sic1 in vivo. These findings reveal an eclipsed transcription-independent role of Whi7 homologs, which is highlighted by genome integrity checkpoints to hold the G1/S transition instantly as a rapid response to unforeseeable replication threats. Key points Whi7 overexpression bypasses the essential function of Mec1 and Rad53 in a transcription-independent way. Whi7 is stabilized by checkpoint-mediated phosphorylation. Whi7 binds and hinders S-CDK-Cks1 from multi-phosphorylation of Sci1, thereby prolonging Sic1 degradation and G1/S transition.

Transcription-independent hold of the G1/S transition is exploited to cope with DNA replication stress