Abstract
The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.
Highlights
The appropriate spatiotemporal regulation of DNA replication is essential to genetic integrity and cell proliferation
Known SLiMs only account for a small fraction (
The prevailing view is that recruitment occurs principally through the ability of the origin recognition complex (ORC) to bind origin DNA in an ATP-dependent manner that depends on DNA encirclement within the central channel of the complex (Bleichert et al, 2018; Li et al, 2018; Sun et al, 2013; Yuan et al, 2017)
Summary
The appropriate spatiotemporal regulation of DNA replication is essential to genetic integrity and cell proliferation. The initiation of DNA replication requires the coordinated action of three proteinaceous factors – the Origin Recognition Complex (ORC), Cdc, and Cdt1 – which coassemble on DNA origins in the G1 phase of the cell cycle to catalyze loading of the Mcm replicative helicase onto chromatin. Five ORC subunits (Orc1-5), as well as Cdc and the six subunits of Mcm, possess an ATPases Associated with diverse cellular Activities (AAA+) domain; Orc and Cdt are the only non-AAA+ proteins used for Mcm loading. S. cerevisiae Cdt possesses a catalyticallyinactive dioxygenase domain at its N-terminus that is necessary for yeast viability and Mcm loading (Frigola et al, 2017; Takara and Bell, 2011); this fold is absent in S. pombe and metazoan Cdt1s
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