Phosphorylation alters the bulk chemical properties of Orc1 to tune DNA binding, phase separation, and heterochromatin partitioning.

<jats:p> The first step in initiating DNA replication is binding of the origin recognition complex (ORC) to chromosomes. Metazoan ORC is recruited to chromatin via the Orc1 intrinsically disordered region (IDR) whose DNA and chromatin binding activity are regulated by Cyclin Dependent Kinase (CDK) phosphorylation. ORC is also enriched in heterochromatin where it is required for the formation and maintenance of a silenced chromatin state. ORC's recruitment to heterochromatin is developmentally and cell cycle regulated but the underlying regulatory mechanism remains unknown. We hypothesized that CDK-dependent phosphorylation of the Orc1 IDR underpins regulated recruitment to heterochromatin. Using bioinformatic analyses, we find that the Drosophila Orc1 IDR (Orc1 <jats:sup>IDR</jats:sup> ) contains an exceptionally high density of CDK phospho-sites and, despite considerable sequence variation, the density of sites, but not their position, is conserved. <jats:italic>In vitro</jats:italic> DNA binding and phase separation experiments reveal that phosphorylation tunes Orc1 <jats:sup>IDR</jats:sup> function in a rheostat-like fashion. Using phospho-mimetic variants, we find that constitutive phosphorylation not only weakens interphase chromatin binding but fully inhibits partitioning of Orc1 <jats:sup>IDR</jats:sup> into heterochromatin. Finally, we use phospho-mimetic variants to probe the importance of site-specific phosphorylation and find that the precise position of sites can be changed provided the new sites are equitably distributed across the sequence. These studies demonstrate that phosphorylation tunes the biochemical properties of the Orc1 IDR to control DNA binding, phase separation, and, consequentially, heterochromatin recruitment. This work suggests that localized dephosphorylation of the DNA binding Orc1 IDR may underlie recruitment of ORC to specific genomic loci. </jats:p>