Cell cycle regulation has shaped replication origins in budding yeast

Chew Theng Lim; Thomas C R Miller; Kang Wei Tan; Saurabh Talele; Anne Early; Philip East; Humberto Sánchez; Nynke H Dekker; Alessandro Costa; John F X Diffley

doi:10.1038/s41594-025-01591-9

Cell cycle regulation has shaped replication origins in budding yeast

Nat Struct Mol Biol. 2025 Jun 30. doi: 10.1038/s41594-025-01591-9. Online ahead of print.

Authors

Chew Theng Lim^#¹, Thomas C R Miller^#^{2

3}, Kang Wei Tan¹, Saurabh Talele^{4

5}, Anne Early¹, Philip East⁶, Humberto Sánchez⁴, Nynke H Dekker^{4

5}, Alessandro Costa⁷, John F X Diffley⁸

Affiliations

¹ Chromosome Replication Laboratory, The Francis Crick Institute, London, UK.
² Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK. tmiller@sund.ku.dk.
³ Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark. tmiller@sund.ku.dk.
⁴ Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
⁵ Department of Physics and Kavli Institute of Nanoscience Discovery, University of Oxford, Oxford, United Kingdom.
⁶ Bioinformatics & Biostatistics, The Francis Crick Institute, London, UK.
⁷ Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK.
⁸ Chromosome Replication Laboratory, The Francis Crick Institute, London, UK. john.diffley@crick.ac.uk.

^# Contributed equally.

PMID: 40588661
DOI: 10.1038/s41594-025-01591-9

Abstract

Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition complex (ORC), Cdc6 and Cdt1 by sequential hexamer loading from two opposed ORC binding sites. Cyclin-dependent kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to the G1 phase. Here, we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MCM-ORC (MO) intermediate). CDK-dependent phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO stabilizes ORC at lower-affinity binding sites required for second hexamer loading. Origins comprising two high-affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers. Strikingly, these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural, strong origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.