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Principal Investigator: MICHAEL R. BOTCHAN
Organization: UNIVERSITY OF CALIFORNIA BERKELEY
Fiscal Year: 2024
Award: $537,835
Funding agency: National Institute of General Medical Sciences
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.
Terms: <ATP phosphohydrolase><ATPase><Address><Adenosine Triphosphatase><Area><Automobile Driving><Behavior><Binding><Biochemical><Biochemistry><Biological Chemistry><Biology><Biophysical Process><Cancers><Cell Body><Cell Communication and Signaling><Cell Cycle Control><Cell Cycle Regulation><Cell Growth in Number><Cell Multiplication><Cell Proliferation><Cell Signaling><Cells><Cellular Proliferation><Chromatin><Chromosomal Organization><Chromosomal Structure><Chromosome Organization><Chromosome Structures><Chromosomes><Complex><DNA><DNA Binding><DNA Binding Interaction><DNA Helicases><DNA Molecular Biology><DNA Replication><DNA Replication Initiation><DNA Synthesis><DNA Unwinding Proteins><DNA biosynthesis><DNA bound><DNA unwinding enzyme><Data><Defect><Deoxyribonucleic Acid><Development><Disease><Disorder><Dwarfism><Elements><Eukaryota><Eukaryote><Event><Exclusion><Gene Expression><Gene Inactivation><Gene Silencing><Genetic><Genetics-Mutagenesis><Genome><Goals><Heterochromatin><Image><In Vitro><Initiation Factor IF-3><Initiation Factors><Intervention><Intervention Strategies><Intracellular Communication and Signaling><Knowledge><Lab Findings><Laboratory Finding><Lead><Liquid substance><Maintenance><Malignant Neoplasms><Malignant Tumor><Membrane><Modeling><Molecular><Molecular Biology><Molecular Interaction><Mutagenesis><Mutagenesis Molecular Biology><Nanism><Nucleic Acids><ORC protein><ORC1><ORC1-Like><ORC1L><ORC1L gene><Oncogenesis><Outcome><Pathology><Pathway interactions><Pb element><Peptide Initiation Factor 3><Peptide Initiation Factor IF-3><Peptide Initiation Factors><Phase><Physical condensation><Probability><Prokaryotic Initiation Factor-3><Prokaryotic Peptide Initiation Factor-3><Property><Proteins><Proteome><Reagent><Replication Initiation><Replication Origin><Role><S Period><S phase><Signal Transduction><Signal Transduction Systems><Signaling><Sorting><Specific qualifier value><Specificity><Specified><Structure><Synthesis Period><Synthesis Phase><System><TIF IF3><Testing><Translation Initiation Factor><Translation Initiation Factor 3><Translational Initiation Factor><Work><biological signal transduction><biophysical mechanism><cell imaging><cellular imaging><condensation><developmental><driving><dwarf><experience><flexibility><flexible><fluid><heavy metal Pb><heavy metal lead><helicase><human disease><imaging><imaging approach><imaging based approach><in vivo><insight><interest><interventional strategy><liquid><malignancy><melting><membrane structure><neoplasm/cancer><novel><ori Region><origin recognition complex><pathway><programs><prospective><protein protein interaction><recruit><social role><structural determinants><structural factors><transcriptional silencing><tumorigenesis>