Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication.

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly. [Display omitted] • ORC1 and CDC6 IDRs have short linear motifs that bind many regulatory proteins • ORC1 and CDC6 have short domains that promote assembly of their AAA+ ATPase domains • ORC1 levels in the cell are controlled by ORC1-ORC1 interactions and by CDC6 • ORC1 and CDC6 form a liquid-liquid phase transition stimulated by DNA Intrinsically disordered regions in the human DNA replication initiation proteins ORC1 and CDC6 contain short protein motifs that interact with multiple cyclin-dependent kinases, a phosphatase, and one another to control cell cycle-regulated initiation of DNA replication, ORC1 intra-nuclear protein levels, and the assembly of their AAA+ ATPase domains. [ABSTRACT FROM AUTHOR]