Studies to Explore DNA Replication Proteins in Functional Assemblies through Intrinsically Disordered Domains

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PROJECT SUMMARY Our long-term goal is to define the molecular mechanisms by which DNA replication is initiated and regulated in metazoans. Cells rely on two AAA+ ATPases, ORC and Cdc6, along with a third factor, Cdt1, to load a latent helicase (the Mcm2-7 complex) as a double hexamer onto replication origins. Upon entering S-phase, Mcm2-7 is activated by the GINS and Cdc45 accessory factors, melting the duplex origin. The resultant CMG (Cdc45/Mcm2-7/GINS) assembly unwinds parental DNA strands and coordinates DNA synthesis by the replisome. Recently, we discovered that metazoan replication initiation factors – specifically the Orc1 subunit of ORC, as well as Cdc6 and Cdt1 – use long, intrinsically disordered regions (IDRs) to bind DNA and partition into liquid phase condensates (LPCs). This and other observations led us to a new functional model for replication, whereby initiator IDRs and LPC propensity controls not only chromatin association, but also Mcm2-7 loading, partner selection, and heterochromatin status. In Aim 1, we will resolve the molecular determinants by which initiator IDRs facilitate condensation. In Aim 2, we will define how initiator IDRs control partner-protein interactions. In Aim 3, we will establish how Orc1 uses its IDR to interface with pericentric heterochromatin through interactions with other LPC-forming proteins such as Hp1. Significant outcomes expected to result from the proposed work include: 1) defining how initiator IDRs – which we have shown to be a novel class of condensate-promoting element – interface with DNA and each other, 2) uncovering new proteins capable of associating with initiation factors, and 3) explaining how ORC connects to the formation and maintenance of genome organization and expression. IDRs have been predominantly thought to serve either as flexible linkers that allow mobility between ordered domains, or as segments that undergo an induced-fit transition into folded structures through protein-protein interactions. Recent work shows that IDRs can fulfill another role in specifying partner-protein interactions and driving the formation of membraneless compartments through liquid phase separation. Our proposal will establish how IDRs can lead to specificity for co-association and potential compartmentalization with origins and other factors. Our efforts will inform areas of molecular biology where IDRs are used to manifest phase-separated compartments or protein/nucleic-acid clustering for functional purposes. As ~25% of proteomes are predicted to be unstructured, the utility of such insights will be broadly significant.