Date of Degree

6-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Amy Ikui

Committee Members

Emmanuel Chang

David Jeruzalmi

Dirk Remus

Duncan Smith

Subject Categories

Cell Biology | Molecular Biology | Molecular Genetics | Structural Biology

Keywords

dna replication, phosphatase, budding yeast, Cdc6, PP2A, mitosis

Abstract

Control of DNA replication is critical for progression of the cell cycle and genomic stability. Cyclin-dependent kinases (CDKs) coordinate numerous phosphorylation events to accomplish two biological tasks for all living organisms: DNA replication and cell division. One CDK, Cyclin-Cdc28, is responsible for cell cycle progression in budding yeast. DNA replication requires a stepwise assembly of the pre-replicative complex on DNA, including Orc1-6, Cdc6, Cdt1 and Mcm2-7, during M-G1 phase. Cdc6 contains eight Cdc28 consensus sites, SP or TP motifs. Clb5-Cdc28 phosphorylates Cdc6-T7 to recruit Cks1, the Cdc28 phospho-adaptor, for subsequent multisite phosphorylation during S phase. There are two phospho-degrons at T39-S43 and T368-S372 which target Cdc6 for SCF-dependent ubiquitylation, followed by proteasome-mediated protein degradation. Cdc6 accumulates in mitosis and is tightly bound by mitotic cyclin Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing. Cdc6-Clb2 binding potentially protects Cdc6 from protein degradation by shielding the N-terminal phospho-degron. These mechanisms ensure high Cdc6 levels in mitosis even when Cdc6 is not bound to DNA.

It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdc28 complex. However, a detailed mechanism for reversing Cdc6 phosphorylation has not been elucidated. Two major phosphatases in yeast, PP2ACdc55 and Cdc14 counteract Cdc28 phosphorylation to regulate the cell cycle. Both have been shown to interact with Cdc6, but the nature of this association is poorly understood. In this thesis, I show that PP2ACdc55 and Clb2 have opposite effects on Cdc6 protein levels. I demonstrate that PP2ACdc55 dephosphorylates the Cdc6-T7 and –T23 sites to release Clb2 from shielding the N-terminal phospho-degron. In collaboration with Mihkel Ӧrd and Mart Loog, we demonstrate that phosphatase Cdc14 dephosphorylates the T368-S372 degron, leading to Cdc6 stabilization in mitosis. Therefore, PP2A-Cdc55 and Cdc14 have distinct target sites in Cdc6, which allows Cdc6 to assemble pre-RCs on DNA in late mitosis. In collaboration with Dirk Remus, we obtained evidence that the Cdc28 inhibitor, Sic1, releases Cdc6 from the inhibitory complex with Clb2·Cdc28·Cks1 to allow Mcm2-7 loading on chromatin. In order to clarify Cdc6 binding dynamics, I collaborated with Andriele Silva and Shaneen Singh. Together, we utilized bioinformatics to generate predicted models of Cdc6 and its binding partners.

Our results suggest that PP2A and Cdc14 sequentially dephosphorylate distinct Cdk1 sites on Cdc6 during mitosis. Sic1 also releases Cdc6 from the inhibitory Clb2·Cdc28·Cks1·Cdc6 complex upon mitotic exit for origin licensing. Such a mechanism ensures faithful once-per-cell-cycle DNA replication to maintain genomic integrity.

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