Science 290, 2309C2312. 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 dephosphorylation upon mitotic exit, increasing ORC1 protein and promoting pre-RC assembly. Cdc6 (Cook et al., 2002; Coverley et al., 2002; Duursma and Agami, 2005; Mailand and Diffley, 2005). Thereafter, during S phase CDC6 is phosphorylated by Cyclin A-CDK2 and re-localizes to the cytoplasm (Delmolino et al., 2001; Jiang et al., 1999; Petersen et al., 2000). In (Vashee et al., 2003). Human ORC1 and CDC6 are also involved directly in regulation of gene expression in mid G1 phase to influence the decision of whether cells will proliferate or not (Hossain and Stillman, 2016). Open in a separate window Figure 1. Dynamic interaction between ORC1 Rabbit Polyclonal to Tau (phospho-Thr534/217) and CDC6 proteins during the human cell cycle.(A) Schematic of dynamic expression pattern of human and the yeast ORC1, CDC6 and Cyclin-CDK kinases across the cell division cycle. (B) Immunoprecipitation of ORC1 from asynchronous U2OS (left panel) and HeLa (right panel) cell lysates showing interactions with CDC6, ORC3, ORC4, Cyclin A and SKP2 proteins. Input and IP are 5% and 30%, respectively. Molecular weight markers, kDa. (C) Dynamic expression profile of pre-RC and cell cycle proteins detected by immunoblotting of extracts from double thymidine block and released synchronized HeLa cells. DNA content is indicated. CYCE and CYCA denotes Cyclin E and Cyclin A, respectively. (D-E) Double thymidine synchronized and released HeLa cell lysate prepared at different time points were immunoprecipitated either with an ORC1 antibody (D) or a CDC6 antibody (E) and immunoblotted as indicated. The input and IgG IP denote loading control and mock IP in the experiment, respectively. It has long been known that Cyclin-CDKs regulate the timing of pre-RC assembly and function, but how they do this in human cells is unclear (Coverley et al., 2002; Li et al., 2004). The activity of Cyclin-CDKs requires their substrates to harbor a specific Cyclin-CDK recognition motif (Cy motif with consensus R/KxL) (Adams et al., 1996; Takeda et al., 2000; Wohlschlegel et al., 2001), although other Cyclin binding motifs have been reported (?rd et al., 2020). The N-terminal regions in both ORC1 and CDC6 harbor the R/KxL type Cy motif as well as multiple CDK phosphorylation sites, and both exist in predicted IDRs of each protein (Figure S1) (Hemerly et al., 2009; Schulman et al., 1998; Wood and Endicott, 2018). The N-terminal regions of yeast Orc1 and Cdc6 also contain predicted IDRs, with no apparent Cy motif in yeast Orc1 (Figure S1). In modelling, CDC6 was docked in between ORC1 and ORC2, consistent with the yeast and ORC-Cdc6 structures (Bleichert et al., 2018; Jaremko et al., 2020; Schmidt and Bleichert, 2020; Tocilj et al., 2017; Yuan et al., 2017). The interaction between GST-CDC6 and MBP-ORC1 was enhanced by ATP (Figures 2A and S2B). Next, many fragments of CDC6 were constructed and binding assays showed that amino acids GSK1324726A (I-BET726) 1C110 within CDC6 were necessary and sufficient to bind to ORC1 protein (Figures 2B, S2C, S2D and S2E). This was surprising since the AAA+ domains of Orc1 and Cdc6 interact in the yeast and ORC-Cdc6_Cdt1-Mcm2-7 (OCCM) complex bound to GSK1324726A (I-BET726) origin DNA and GSK1324726A (I-BET726) ORC stimulates the ATPase activity of Cdc6 (Randell et al., 2006; Schmidt and Bleichert, 2020; Speck and Stillman, 2007; Yuan et al., 2017). Nevertheless, the 1C110 region of GSK1324726A (I-BET726) GST-CDC6 bound to MBP-ORC1 protein while the AAA+ containing 110C560 fragment of Cdc6 did not bind (Figures 2B and S2E). Moreover, GSK1324726A (I-BET726) internal deletions of small N-terminal regions (11C20, 21C30, 51C70 and 71C90) within full length GST-CDC6 protein still.