A new class of disordered elements controls DNA replication through initiator self-assembly.

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• DNA replication initiation in metazoans occurs at numerous chromosomal origins.
• The Origin Recognition Complex (ORC), Cdc6, and Cdt1 load the Mcm2-7 helicase at origins.
• Current models view replication origins as autonomously functioning and linearly arranged.

Purpose of the Study:

• To investigate the organization and communication between replication origins.
• To explore the in vitro behavior of the DNA replication initiation machinery.
• To identify novel mechanisms regulating DNA replication initiation.

Main Methods:

• Investigated the behavior of *D. melanogaster* replication initiation factors (ORC, Cdc6, Cdt1) in vitro.
• Utilized techniques to observe liquid-liquid phase separation (LLPS) upon DNA binding.
• Analyzed the role of intrinsically disordered regions (IDRs) in driving LLPS and initiator functions.

Main Results:

• The replication initiation machinery (ORC, Cdc6, Cdt1) undergoes LLPS when binding DNA.
• Intrinsically disordered regions (IDRs) in these initiators drive LLPS and form a new class of phase-separating elements.
• Initiator IDRs regulate chromosome recruitment, co-assembly, and Mcm2-7 helicase loading.

Conclusions:

• LLPS of replication initiators provides a mechanism for organizing replication origins.
• This higher-order organization helps explain how CDK activity controls replication initiation.
• Replication programs may involve inter-origin communication and organization beyond linear models.