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

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.

Grant Number: 5R01GM141045-04
NIH Institute/Center: NIH
Principal Investigator: MICHAEL BOTCHAN