(A) Analysis of tRFP-Cdc6 (2.5 µM) and eGFP-Cdt1 (2.5 µM) recruitment to pre-formed ORC/Cy5-dsDNA (2.5/2.5 µM) droplets in the absence (‘-ATP’) and presence (‘+ATP’) of ATP (1 mM). (B) eGFP-Cdt1 (500 nM) or eGFP-FUS (500 nM) was added to reactions and assessed for the ability to co-localize with preformed ORC/Cy5-dsDNA droplets (2.5/2.5 µM). In samples containing eGFP-Cdt1, all ORC/Cy5-dsDNA droplets are enriched for eGFP signal. No enrichment is observed in samples containing eGFP-FUS.). (C) Quantitation of eGFP signal intensity within and outside of ORC/Cy5-dsDNA droplets for samples in panel (B). (D) Schematic of a depletion assay to assess Mcm2-7 phase partitioning; both the supernatant and pellet were assessed for either the loss or enrichment, respectively, of Mcm2-7. (E) Mcm2-7 (500 nM) was not depleted from the dilute phase and, consistently, was absent from the condensed phase (F). (G) Loading reactions were prepared that contained either ORC, Cdc6, and Cdt1 (OCC) or ATP, or both the OCC and ATP, and phase separation of Mcm2-7 was assessed by the depletion assay. In the presence of both the OCC and ATP, Mcm2-7 is significantly enriched in the condensed phase. (H) Loading reactions were set up that contained both ATP and DNA, but from which individual initiators were removed (ORC, Cdc6, or Cdt1), and the depletion assay performed. In the absence of any one initiator, Mcm2-7 no longer partitions to the pelleted phase.