biostudies-literatureSosenko Piscitello ASwiss National Science FoundationVetenskapsrådetThe Mark Foundation for Cancer ResearchKarolinska InstitutetCancerfonden8268https://www.ebi.ac.uk/biostudies/studies/S-EPMC12432183biostudies-literatureUnknown16(1)Cyclin-dependent kinases (CDKs) coordinate DNA replication and cell division, and play key roles in tissue homeostasis, genome stability and cancer development. The first step in replication is origin licensing, when minichromosome maintenance (MCM) helicases are loaded onto DNA by CDC6, CDT1 and the origin recognition complex (ORC). In yeast, origin licensing starts when CDK activity plummets in G1 phase, reinforcing the view that CDKs inhibit licensing. Here we show that, in human cells, CDK4/6 activity promotes origin licensing. By combining rapid protein degradation and time-resolved EdU-sequencing, we find that CDK4/6 activity acts epistatically to CDC6 and CDT1 in G1 phase and counteracts RB pocket proteins to promote origin licensing. Therapeutic CDK4/6 inhibitors block MCM and ORC6Nature communicationsTemporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics.PMC12432183182487186230Lemmens BHawgood MNtallis SGBartek JUrien BNilsson ASGiglio GBajgain PHalazonetis TDSosenko Piscitello ASayyid AHDionellis VSfalseTemporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics.Cyclin-dependent kinases (CDKs) coordinate DNA replication and cell division, and play key roles in tissue homeostasis, genome stability and cancer development. The first step in replication is origin licensing, when minichromosome maintenance (MCM) helicases are loaded onto DNA by CDC6, CDT1 and the origin recognition complex (ORC). In yeast, origin licensing starts when CDK activity plummets in G1 phase, reinforcing the view that CDKs inhibit licensing. Here we show that, in human cells, CDK4/6 activity promotes origin licensing. By combining rapid protein degradation and time-resolved EdU-sequencing, we find that CDK4/6 activity acts epistatically to CDC6 and CDT1 in G1 phase and counteracts RB pocket proteins to promote origin licensing. Therapeutic CDK4/6 inhibitors block MCM and ORC62025-01-01T00:00:00Z2025 Sep2026-06-02T08:46:23.126Z2026-04-16T03:12:57.673ZS-EPMC124321834094032610.1038/s41467-025-63669-8