Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

Abstract

The initiation of DNA replication involves the 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 in ORC1 and CDC6, including Cyclin-binding (Cy) motifs, mediate Cyclin-CDK dependent and independent protein-protein interactions, conditional on cell cycle phase. The ORC1 Cy motif mediates an auto-regulatory self-interaction, and the same Cy motif prevents CDC6 binding to ORC1 in mitosis, but then facilitates the destruction of ORC1 in S phase. In contrast, in G1, the CDC6 Cy motif promotes ORC1-CDC6 interaction independent of Cyclin-CDK protein phosphorylation. CDC6 interaction with ORC also requires a basic region of ORC1 that in yeast mediates ORC-DNA interactions. We also demonstrate that protein phosphatase 1 binds directly to a SLiM in ORC1, causing de-phosphorylation upon mitotic exit. Thus, Cy-motifs have wider roles, functioning as a ligand and as a degron.

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

“…In line with this idea, we show that human Cdt1, like Dm Cdt1, forms condensates in a DNA-dependent manner (Figure 2—figure supplement 1G). A recent report demonstrating an essential function for the phosphorylation-regulated conversion of intramolecular IDR interactions in human Orc1 to intermolecular interactions between ORC and Cdc6 IDRs (Hossain et al, 2019) is similarly supportive with such a proposal (Figure 6). We anticipate that these interactions, possibly augmented by other transient IDR-IDR interactions forming in both cis and trans , facilitate some form of condensation or localized clustering of initiation factors for the functional assembly of pre-RCs.…”

Distinct and sequential re-replication barriers ensure precise genome duplication

“…The fact that Cdt1-Cy has the highest activity of all the variants tested here may be a reflection of that additional negative regulation in the so-called “PEST domain.” In our experiments, Cdt1 chromatin binding is barely detectable which is consistent with the transient Cdt1 residence at origins during budding yeast MCM loading in vitro [ 68 , 73 ]. Alternatively, the Cy motif mutation may disrupt more than only Cyclin binding such as has been recently reported for ORC [ 74 ]. Clearly the spectrum of Cdt1 biological activities can be tuned by combinations of phosphorylations and dephosphorylations, and continued in-depth analyses will yield additional insight into Cdt1 regulation and function.…”

The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

“…Interestingly, we found there were functionally essential regions in the lesser conserved IDR regions of both ORC1 and ORC2. Our lab has recently reported that a Cyclin-motif bearing region of ORC1 (180 - 240 aa) is essential for binding to CDC6 at the right time during the cell cycle to enable pre-RC formation (Hossain et al, 2019). And similarly, based on homology, a previously identified putative NLS motif (Lidonnici et al, 2004) also had hits nominating that region as essential in our ORC1 screen (Figure 1f).…”

“…The known functional domains in ORC1, including the BAH, AAA+ and WHD were identified using the open reading frame tiling CRISPR-Ca9 sgRNA screen, as well as other regions of ORC1, including the intrinsically disordered region (IDR; amino acids 180-480, Figure 1e) which we know binds Cyclins E and A-CDK2 and CDC6 (Hossain et al, 2019) as well as many other proteins we have identified and are characterizing in detail. The screen also identified an essential region of ORC1 in and around amino acid 750-790 (Figure 1a-b) which may represent the pericentrin- AKAP450 centrosomal targeting (PACT) domain that localizes ORC1 to centrosomes to regulate correctly centrosome and centriole copy number (Hemerly et al, 2009).…”

“…In ORC2, multiple, essential domains were identified, including the AAA+-like domain and the WHD. The WHD of human ORC2 controls access of human ORC to DNA by inserting itself into the DNA binding channel prior to activation of the protein by binding of ORC1 and subsequent binding of CDC6 (Hossain et al, 2019; Jaremko et al, 2020). The ORC2-carboxy terminus binds to ORC3 and ORC2 is also known to bind to PLK1, the mitotic protein kinase (Song et al, 2011).…”