The internal environment of a cell is compartmentalized both by membrane-bound organelles and by protein-rich and protein/nucleic acid-rich compartments that lack an enclosing membrane. These membraneless compartments are commonly called biomolecular condensates and form through an ability of their constituent factors to undergo liquid–liquid phase separation (LLPS) (reviewed in Banani et al., 2017). Cellular bodies that form by LLPS are often spherical due to surface tension minimization across the phase boundary (Elbaum-Garfinkle, 2019). Sphericity, however, is not a defining feature of biomolecular condensates and many variables can contribute to droplet (de)formation, including the physical parameters of the liquid phase (e.g., surface tension, viscosity, and size) and external variables (e.g., application of a force and surface interactions). Nonspherical condensates often nucleate from or assemble along relatively rigid intracellular scaffolds. This effect is seen with cellular signaling complexes (Banjade and Rosen, 2014; Li et al., 2012; Su et al., 2016) and tight junctions (Beutel et al., 2019), which spread with the dimensions of the plasma membrane. Likewise, the chromatin-associated synaptonemal complex (Rog et al., 2017) and the perichromosomal layer (Booth and Earnshaw, 2017) are predicted to form a protein-rich phase that contours with the more rigid nature of their substrate, mitotic chromosomes (Batty and Gerlich, 2019; Goloborodko et al., 2016; Houlard et al., 2015; Sun et al., 2018).

We previously discovered that the factors responsible for initiating DNA replication in metazoans, in particular the fly homologs of the origin recognition complex (ORC) and Cdc6, as well as fly and human Cdt1, undergo DNA-dependent LLPS at physiological salt and protein concentrations in vitro (Parker et al., 2019). Recent studies have also demonstrated that human Orc1 and Cdc6 can phase separate in the presence of DNA (Hossain et al., 2021). In live cells, initiators first associate with chromatin in mitosis where they appear to uniformly coat, or ‘wet’, anaphase chromosomes (Baldinger and Gossen, 2009; Kara et al., 2015; Parker et al., 2019; Sonneville et al., 2012). These cytological observations are consistent with expectations for a protein that undergoes a condensation reaction with a relatively rigid intracellular scaffolding partner. The ability to condense on DNA is conferred by a metazoan-specific, N-terminal intrinsically disordered region (IDR) present in Orc1, Cdc6, and Cdt1. For Orc1, this region is required for binding chromatin in tissue culture cells and for viability in flies (Parker et al., 2019). In vitro assays show that the Orc1 IDR is dispensable for ATP-dependent DNA-binding by Drosophila melanogaster ORC and loading of the Mcm2–7 replicative helicase (Schmidt and Bleichert, 2020). However, at physiological ionic strength and protein concentrations, initiator IDRs drive initiator coalescence into a condensed phase that both supports ATP-dependent recruitment of Mcm2–7 and can be controlled by CDK-dependent phosphorylation (Parker et al., 2019), an event that directly regulates initiator mechanism (Findeisen et al., 1999; Lee et al., 2012). Together, the available data suggest that LLPS facilitates the formation of an enriched layer of initiation factors that coats mitotic chromosomes to enhance the kinetics of helicase loading, a process that is completed within a relatively narrow window of the eukaryotic cell cycle (Dimitrova et al., 2002; Méndez and Stillman, 2000; Okuno et al., 2001).

How metazoan initiator IDRs facilitate DNA-dependent LLPS at a molecular level is unknown. Protein multivalency underlies the formation of intermolecular interaction networks that can drive LLPS. In many phase-separating systems, functional multivalency is contributed by the presence of one or more IDRs (Li et al., 2012). Although phase-separating IDR sequences vary between systems, some sequence features capable of supporting LLPS have begun to emerge. For example, many phase-separating IDRs have low amino acid sequence complexity (so-called low complexity domains [LCDs]) and are highly enriched for a few select amino acids (e.g., the nucleolar protein FIB1 [Feric et al., 2016], the P-granule protein Laf-1 [Elbaum-Garfinkle et al., 2015], the stress granule protein hnRNPA1 [Molliex et al., 2015], the transcription factor EWS [Chong et al., 2018], and the transcriptional coactivator MED1 [Sabari et al., 2018]). However, there exist important exceptions to this correlation, including phase separation by the intrinsically disordered nephrin intracellular domain (NICD) (Pak et al., 2016) and by metazoan replication initiators (Parker et al., 2019), both of which possess high sequence complexity IDRs. Thus, LCDs are but a subtype of phase-separating IDR.

Literature reports of phase-separating sequences are increasing rapidly, yet relatively few of these studies define the molecular basis for LLPS. An important goal of the field is to understand and ultimately predict the extent to which different disordered protein regions utilize a generally shared or individually distinctive set of cohesive interactions to drive phase separation. Interestingly, many condensates (Table 1) dissolve upon treatment with the aliphatic alcohol 1,6-hexanediol (1,6-HD), a compound that permeabilizes the nuclear pore by disrupting weak hydrophobic Phe-Gly (‘FG’)-repeat interactions that are prevalent in the assembly (Patel et al., 2007; Shulga and Goldfarb, 2003). The broad sensitivity of protein LLPS to 1,6-HD could be taken to indicate that phase separation mechanisms are generalizable, at least for certain IDR classes (Kroschwald et al., 2017). Consistently, IDR sequence aromaticity has been shown to be an essential feature in a variety of phase-separating systems (Chiu et al., 2020; Chong et al., 2018; Lin et al., 2017; Nott et al., 2015; Pak et al., 2016; Qamar et al., 2018; Wang et al., 2018). Nevertheless, other interaction types appear to predominate in other LLPS systems. For example, charged residues can facilitate phase separation either through homomeric protein–protein interactions (Elbaum-Garfinkle et al., 2015; Mitrea et al., 2018; Nott et al., 2015) or through heteromeric protein–protein (Ferrolino et al., 2018; Pak et al., 2016) and protein–nucleic acid interactions (Lin et al., 2015; Zhang et al., 2017). We note also that membraneless organelles in vivo have a particular compositional bias that often underpins their utility, implying that there exists a molecular ‘grammar’ or ‘sorting code’ that accepts a particular subset of partner factors while excluding inappropriate factors. Consistent with this idea, we have observed that initiator IDRs can corecruit pathway-specific partner proteins but not other types of phase-separating sequences (such as human Fused in Sarcoma [FUS]) (Parker et al., 2019). An understanding of the molecular rules that govern IDR sorting mechanisms is still in its infancy.

Here, we use D. melanogaster Cdt1 as a model to dissect the molecular basis for DNA-dependent LLPS by metazoan replication initiator proteins. We find that phase separation by Cdt1 is unaffected by treatment with 1,6-HD and that sequence aromatics, which represent <3% of the total initiator IDR amino acids, are dispensable for condensate formation. We show that protein–DNA interactions are electrostatic in nature and contribute an adhesive force to LLPS, with DNA functioning as a counterion bridge. These interactions synergize with cohesive intermolecular IDR–IDR interactions, which are driven by the hydrophobic effect. Using mutagenesis, we demonstrate that intermolecular IDR–IDR interactions serve as the primary driving force behind initiator phase separation, with DNA-mediated bridging interactions playing a secondary but still facilitative role. Collectively, these studies provide a detailed picture of the biochemical mechanisms underlying IDR LLPS in replication initiation factors and reinforce the concept that biomolecular condensates can form by a variety of different molecular interaction types.

In previous work, we discovered that several factors used to initiate DNA replication in metazoans – in particular the Orc1 subunit of ORC, as well as Cdc6 and Cdt1 – possess an N-terminal IDR that promotes protein phase separation upon binding DNA (Parker et al., 2019). When these initiator proteins are combined and DNA is added, they form comingled condensates that exclude noncognate phase-separating proteins and are active for the ATP-dependent recruitment of the Mcm2–7 helicase. The biophysical mechanisms underlying LLPS by replication initiators have remained unknown.

Here, we have used Drosophila Cdt1 as a model system to dissect the molecular basis for DNA-dependent phase separation by initiation factors. Our studies show that this condensation mechanism has several distinctive features compared to other proteins characterized to date that also undergo LLPS. We find that two types of intermolecular interactions synergize with each other to drive phase separation by Cdt1. One set of interactions occur between Cdt1 and DNA. These associations appear to be nonspecific and electrostatic in nature, with DNA serving as a polyanion that bridges the cationic IDRs of multiple Cdt1 molecules (Figures 2 and 5G). A second set of interactions that occur between the IDRs of different Cdt1 protomers were also identified; these appear to be salt-insensitive and primarily hydrophobic in nature (Figures 4 and 5G). Importantly, we find that inter-IDR interactions can drive Cdt1, ORC, and Cdc6 LLPS in the absence of DNA (Figure 3 and Figure 4—figure supplement 1). When we abolish these interactions in Cdt1, DNA is insufficient to induce phase separation (Figure 5). Thus, inter-IDR interactions provide the primary force behind initiator LLPS, with electrostatic DNA-bridging interactions contributing an additional adhesive force.

In the absence of a crowding reagent, DNA is required to induce initiator phase separation at physiological salt and protein concentrations (Parker et al., 2019). This dependency suggests that DNA serves as an essential nucleating factor that determines where Cdt1 can form condensates, ensuring that initiator LLPS is restricted to a chromatin context. Interestingly, other anionic biopolymers, such as RNA and heparin, are also capable of inducing phase separation by Cdt1. This type of scaffold ‘promiscuity’ has been observed in noninitiator systems that undergo LLPS, such as for the nucleolar protein fibrillarin (Feric et al., 2016). Given that the cytosol contains multiple anionic biopolymers, future work will need to answer how replication initiators are specifically targeted to chromatin, a question that is similarly relevant for RNA-dependent cellular bodies (Berry et al., 2015; Elbaum-Garfinkle et al., 2015; Feric et al., 2016; Guillén-Boixet et al., 2020; Mitrea et al., 2016; Molliex et al., 2015; Van Treeck et al., 2018). The selection of an appropriate nucleic acid scaffold across different phase-separating systems is likely to rely on both general mechanisms, such as the sequestration of alternative, inappropriate scaffolds by proteins with different or nonexistent LLPS properties, and by system-specific mechanisms, such as a requirement for particular sequences or secondary structure (Langdon et al., 2018; Maharana et al., 2018).

A growing body of work has demonstrated the importance of aromatic residues in driving protein phase separation (Chiu et al., 2020; Chong et al., 2018; Lin et al., 2017; Nott et al., 2015; Pak et al., 2016; Qamar et al., 2018; Wang et al., 2018), along with a related role for π–π and π–cation interactions (Vernon et al., 2018). In parallel, many studies have shown that condensates formed through aromatic residue interactions are readily dissolved by 1,6-HD (Table 1). Cdt1 LLPS is both resistant to treatment with 1,6-HD and does not rely on aromatic residues within its IDR for phase separation (Figure 1). ORC and Cdc6 phase separation is also resistant to 1,6-HD (Figure 1—figure supplement 2). Although we have not explicitly investigated the role of aromatic residues within these proteins, we note that the fraction aromatic residues of Orc1 (0.019) and Cdc6 (0.012) is lower than that of Cdt1 (0.027), suggesting that aromaticity is dispensable for LLPS by replication initiation factors in general. Similarly, charged residues have an established role in facilitating homomeric inter-IDR interactions that lead to phase separation (Elbaum-Garfinkle et al., 2015; Mitrea et al., 2018; Nott et al., 2015), yet the inter-IDR interactions that drive initiator phase separation (Figure 4 and Figure 4—figure supplement 1) are salt insensitive.

Instead of relying on aromatic-mediated π–π/π–cation interactions or charge–charge interactions, our data show that interactions between Cdt1 IDRs are instead facilitated by branched hydrophobic residues. Although hydrophobic sequences have been shown to be important for the phase separation of other factors, such as the extracellular matrix protein elastin (Quiroz and Chilkoti, 2015; Reichheld et al., 2017; Yeo et al., 2011) and the NICD (Pak et al., 2016), important mechanistic distinctions exist with respect to metazoan replication initiators. For example, both NICD and Cdt1 bear an identical FCRs (FCR = 31%) and rely on a counterion for phase separation. However, inter-IDR interactions within NICD appear to rely primarily on sequence aromatics (11% aromatic content), with hydrophobic residues playing only a supporting role in the self-association of this protein (Pak et al., 2016). By contrast, elastin is more like metazoan replication initiators in that it is relatively devoid of aromatic residues (3.9%) and contains a similar content of branched hydrophobic amino acids that underly phase separation (elastin and Cdt1 fraction-L,I,V = 0.21 and 0.19, respectively); however, elastin also differs from initiators in that it readily forms condensates in the absence of a counterion. Elastin’s hydrophobic sequences are additionally contained within highly repetitive ‘VPGVG’ and ‘GLG’ sequences, and scrambling or shuffling these motifs negatively impacts elastin phase separation (Pepe et al., 2008; Pepe et al., 2005; Toonkool et al., 2001). For their part, initiator IDRs lack such repeats and (for Cdt1 at least) LLPS is insensitive to sequence order. We speculate that the inter-IDR interactions which occur in initiators are weaker than those of Elastin due to the nonrepetitive, distributed nature of their hydrophobic residues, a property that necessitates additional counterion-mediated interactions to promote Cdt1 self-assembly.

Understanding the distinctive molecular interactions of different phase-separating systems is essential for developing in silico approaches for predicting the cellular partitioning of a given IDR from primary sequence alone. Such efforts will ultimately require an understanding of both the specific and general features of condensates’ scaffolding components (e.g., repetitive motifs vs. amino acid composition). The present work highlights the importance of the general sequence features of initiator IDRs (as opposed to strict amino acid order) in promoting LLPS. Strikingly, we find that the Cdt1 IDR can be fully scrambled without losing the ability to form condensates (Figure 3I, J) and that condensates formed from the scrambled variant can recruit wild-type Cdt1 into droplets (Figure 3—figure supplement 3). Regions of NICD can similarly be scrambled without losing the ability to form cellular condensates (Pak et al., 2016), as can the LCDs of certain transcription factors without affecting promoter targeting (Brodsky et al., 2020). These studies demonstrate the importance of sequence composition in not only driving phase separation but also in facilitating interactions with partner proteins, and is consistent with recent work showing that proteins with similar functional annotation have disordered sequences with similar, evolutionary conserved physical–chemical features (pI, composition, FCR, kappa, etc.) (Zarin et al., 2019). Our observations involving the cocondensation properties of Cdt1, ORC, and Cdc6 – three proteins with IDRs of similar amino acid composition but no direct sequence homology (Parker et al., 2019) – provide experimental support for this ‘like-recruits-like’ concept. Future work will need to address whether heteromeric inter-IDR interactions (e.g., Orc1^IDR–Cdc^IDR) are indeed governed strictly by sequence composition or whether linear sequence motifs facilitate specific interactions, as was recently suggested for human Orc1 and Cdc6 (Hossain et al., 2021). Further, while metazoan Cdt1 orthologs have no identifiable sequence identity across their disordered domains (Figure 5—figure supplement 2A, B), they do have a similar amino acid composition (Parker et al., 2019). This led us to predict that the capacity to phase-separate was likely conserved across metazoan initiators and we confirmed this for human Cdt1 (Parker et al., 2019). These predictions were further validated for human Orc1 and Cdc6 with the demonstration that these proteins form condensates in the presence of DNA (Hossain et al., 2021). These results suggest that certain classes of disordered domains can have conserved functionality in the absence of linear sequence identity.

Although amino acid composition is the primary determinant underpinning LLPS by replication initiators, short sequence motifs can nonetheless play a critical role in modulating condensation behavior and controlling biological function. Sequence information within the Orc1 and Cdt1 IDR’s is high, and while saltatory leaps seem to have occurred between phyla (Figure 5—figure supplement 2B), the conservation of sequence homology within the Drosophila genus (Figure 5—figure supplement 2C and Parker et al., 2019), over millions of years, is remarkable and indicates an evolutionary pressure on sequence order for unknown function. A conserved sequence feature within the IDRs of Orc1, Cdt1, and Cdc6 are CDK phosphorylation sites which are known to regulate replication initiation in vivo (Findeisen et al., 1999; Lee et al., 2012). We have previously shown that the phosphorylation of these sites – which are distributed throughout the initiator IDRs – by CDKs abrogates the ability of the Drosophila initiators to form condensates (Parker et al., 2019), and a recent finding shows that phosphorylation impedes inter-IDR interactions between human ORC1 and CDC6 as well (Hossain et al., 2021). Notably, consensus motifs for CDK-dependent phosphorylation (full site = [S/T]PX[R/K] and minimal site = [S/T]P) are conserved in a majority of metazoan Cdt1s (Figure 5—figure supplement 2D) and in all sequenced Drosophilidae Cdt1 orthologs (Figure 5—figure supplement 2E), leading us to predict that phospho-tunable phase separation is a broadly conserved mechanism for regulating metazoan replication licensing. Given that a scrambled IDR can still support LLPS in vitro through both self-interactions and cross-interactions with a wild-type IDR sequence (Figure 3), it will be useful to determine whether such a construct (with or without the native CDK sequences) can support normal kinetics of chromatin association and MCM recruitment and/or cell viability. Such studies, along with deletion and more targeted mutagenesis efforts, will be important to define how the plasticity of IDR sequences can be fine-tuned to elicit specific timing and partnering responses.

Defining the molecular ‘grammar’ that encodes LLPS in metazoan initiators likewise has implications for understanding the impact of initiator phase separation in vivo. Admittedly, neither this nor our previous study (Parker et al., 2019) provides unequivocal evidence for initiator phase separation in cells. It is also unclear what function such concentrated initiator assemblies might have, such as improving the kinetics or efficiency of helicase loading (as we have suggested here and previously [Parker et al., 2019]), or something else entirely, such as maintaining the appropriate nuclear levels of the licensing factors (as proposed in Hossain et al., 2021). We note that our system lacks the convenience of identifying initiator condensation morphologically (i.e., by the presence of round cellular foci), as mitotic chromosomes – the stage of initiator assembly – are relatively rigid structures built from a central proteinaceous scaffold through an energy-consuming reaction (Earnshaw and Laemmli, 1983; Gibcus et al., 2018; Hirano and Mitchison, 1994; Kim et al., 2020). Thus, we predict that initiators condense on the surface of mitotic chromosomes without altering their shape. Consistently, work in multiple model organisms has revealed a switch-like transition in initiator localization during anaphase, at which point initiators begin to coat chromosomes (Baldinger and Gossen, 2009; Kara et al., 2015; Parker et al., 2019; Sonneville et al., 2012). We predict that phosphorylation-dependent control of initiator self-assembly underlies this behavior and produces a dense, replication-competent zone of chromatin-bound initiators poised to drive the precipitous, genome-wide loading of the Mcm2–7 complex in late mitosis (Dimitrova et al., 2002; Méndez and Stillman, 2000; Okuno et al., 2001). This moment in the cell cycle is an opportune time to prepare for replication, as conflicts with the transcriptional machinery are minimized (Palozola et al., 2017) and the chromatin substrate is, relative to interphase, uniformly compacted (Ou et al., 2017). The identification of Cdt1 mutants that selectively block phase separation, but not DNA binding (Figure 5), affords an opportunity to directly investigate the physical nature of these global initiator interactions in vivo.

Beyond helicase loading and licensing, initiator LLPS might also serve to prime replisome assembly. Following helicase loading, Mcm2–7 is phosphorylated by the Dbf4-dependent kinase, a heterodimer of Cdc7 and Dbf4 (Deegan et al., 2016; Sheu and Stillman, 2010; Yeeles et al., 2015). Intriguingly, the Drosophila homolog of Dbf4, Chiffon, contains multiple IDRs, including a large internal IDR (residues 903–1209) that possesses striking compositional similarity to initiator-type IDRs (Figure 5—figure supplement 3). The identification of an IDR in Chiffon with compositional similarity to the initiator-type IDRs suggests that such sequences may be present in chromatin-associated factors beyond replication licensing components. Indeed, the capacity to undergo DNA-dependent phase separation may have broad utility for chromatin-localized factors and their respective cellular pathways. Thus, our future work will aim to develop algorithms that accurately identify proteins proteome wide that possess disordered domains with compositional homology to initiator IDRs and to understand how these sequences impact protein functional dynamics.

In summary, the present work provides a detailed view of the DNA-dependent LLPS mechanism for Drosophila Cdt1. Due to a high level of IDR compositional homology and an ability to form comingled phases (Parker et al., 2019), the mechanism we describe for Cdt1 phase separation likely extends to the other replication initiation factors, ORC and Cdc6. These studies set the stage for investigating the physiological significance of initiator self-assembly in replication licensing and for identifying other sequences in the proteome that possess IDRs capable of associating with chromatin and initiation factors alike.

All data generated during this study is included in the manuscript and supporting files.

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Author details

1. Matthew W Parker

   Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Jonchee A Kao and Alvin Huang

   For correspondence

   matthew.parker@utsouthwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7571-0010

2. Jonchee A Kao

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Matthew W Parker and Alvin Huang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1701-3265

3. Alvin Huang

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Matthew W Parker and Jonchee A Kao

   Competing interests

   No competing interests declared

4. James M Berger

   Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-0666-1240

5. Michael R Botchan

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Funding acquisition, Methodology, Supervision, Writing – original draft, Writing – review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-0459-5518

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