Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14, and Sic1 for origin licensing in S. cerevisiae.

Jasmin Philip,Mart Loog,Mihkel Örd,Amy E Ikui,Andriele Silva,John Fx Diffley,Dirk Remus,Shaneen Singh

doi:10.7554/elife.74437

Abstract

Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In Saccharomyces cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, Cdk1 inhibitor Sic1 releases Clb2·Cdk1·Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing are promoted by phosphatases through the attenuation of distinct Cdk1-dependent Cdc6 inhibitory mechanisms.

Highlights

Pre-replicative complexes are assembled on DNA to license replication origins in M–G1 phase
These results indicate that PP2ACdc55 and Clb2 regulate Cdc6 protein levels in a contradictory manner
We show that PP2ACdc55 dephosphorylates Cdc6 at T7 and T23 to disrupt the Cdc6–Clb2 complex in mitosis

Summary

Introduction

Pre-replicative complexes (pre-R Cs) are assembled on DNA to license replication origins in M–G1 phase. The pre-RC components are recruited in a sequential fashion that starts with the origin recognition complex (Orc1–6) followed by Cdc Cdt, which eventually load the Mcm helicase on DNA (Bell and Stillman, 1992; Santocanale and Diffley, 1996; Newlon, 1997; Labib et al, 2001; Tanaka and Diffley, 2002). At the onset of S phase, cyclin-dependent kinase (Cdk1) phosphorylates pre-RC components such as Cdc which prevents reinitiation of DNA replication through multiple mechanisms (Nguyen et al, 2001; Wilmes et al, 2004). Cdc phospho-d egrons are created by Cdk, which directs Cdc protein degradation via SCF-m ediated ubiquitination in Saccharomyces

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