Molecular determinants of phase separation for Drosophila DNA replication licensing factors

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Abstract
Liquid–liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.
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Funding Information
  • National Institute of General Medical Sciences (R01GM141045-01)
  • National Cancer Institute (R01CA030490)
  • National Institute of General Medical Sciences (F32GM116393)
  • UC Berkeley Jessie Rabinowitz Award
  • Cancer Prevention and Research Institute of Texas (RR200070)
  • Welch Foundation (I-2074-20210327)

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