Plant Molecular and Cellular Biology Lecture 8: Mechanisms of Cell Cycle Control and DNA Synthesis Gary Peter

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1 Plant Molecular and Cellular Biology Lecture 8: Mechanisms of Cell Cycle Control and DNA Synthesis Gary Peter 9/10/2008 1

2 Learning Objectives Explain why a cell cycle was selected for during evolution Explain the conceptual basis for cell cycle regulation Explain the structure and function of the proteins that mediate cell cycle progression 9/10/2008 PMCB Lecture 9: G. Peter 2

3 Why Cell Cycle Control Why was a cell cycle selected for? Way to control cell size Coordinate/control growth and development Insure environment is favorable for replication and division and that each of the stages is completed prior to moving on to the next stage Series of checkpoints insure completion of critical steps before progressing forward in cell cycle 9/10/2008 PMCB Lecture 9: G. Peter 3

4 Genetic Strategy for Dissecting the Cell Cycle Forward genetic approaches in Sacchromyces cerevisiae and Schizosacchromyces pombe were critical to identifying genes control cell cycle progression Broad approach screening for cell division cycle mutants Obtain collection of temperature sensitive lethals (nitrosoguanine) Screen collection with time lapse photomicroscopy 9/10/2008 PMCB Lecture 9: G. Peter 4

5 Genetic Dissection of the Cell Cycle: Temperature Sensitive Mutants Conditional mutants (temperature sensitive) isolated where cells arrest at specific cell cycle stages Propagated at permissive temperature, Restrictive temperature cycle progresses till the step at which mutant protein is needed for progression 9/10/2008 PMCB Lecture 9: G. Peter 5

6 Genetic Analysis of Cell Division Cycle in S. cerevisiae Definition of a cell division cycle (cdc) mutant Mutants that arrest cell division at a unique point no matter what stage of the cycle they were at when transferred from permissive to nonpermissive temperature The fact that all cdc mutants arrested with the same motherbud morphology facilitated this 9/10/2008 screening PMCB Lecture 9: G. Peter 6 Biochem. Molec Bio. of Plants, 2000

7 Genetic Analysis of Cell Division Cycle in S. cerevisiae 10% of all ts mutants are cdc suggesting about 500 genes are involved; however only 70 were easily identified with this approach why? Other approaches for making mutations has identified more cdc genes Hartwell, 1991 Genetics 129: /10/2008 PMCB Lecture 9: G. Peter 7

8 Eukaryotic Cell Division Cycle: A Conserved, Dependent Pathway of Stages and Checkpoints Interphase G1- Growth S DNA synthesis G2- Growth Mitosis M- Nuclear division M- Cytokinesis G o specialized resting state prior to entry into G1 9/10/2008 PMCB Lecture 9: G. Peter 8

9 Molecular Analyses of this Collection of Mutants Suggests Number of Themes The role of transcriptional control for entry into the cell cycle The central role of post translational regulation Protein kinases and protein phosphatases in cell cycle progression The importance of ubiquitin mediated or targeted proteolysis The role of protein:protein interactions 9/10/2008 PMCB Lecture 9: G. Peter 9

10 Protein Kinases Regulate Cell Cycle Progression Cyclin dependent kinases (cdks) are the critical regulators Kinase activity rises and falls during the cell cycle Phosphorylation of intracellular proteins by cdks activates these proteins to initiate cell cycle progression Cdks are involved in the main feedback loops that control progression In contrast to yeast (cdc2 or cdc28), higher eukaryotes have multiple cdks that are specific for various cyclins and stages of the cell cycle: Two interact with G1 cyclins One with G1/S cyclins One with M cyclins 9/10/2008 PMCB Lecture 9: G. Peter 10

11 Cyclins: Conserved Activators of Cyclin Dependent Kinases Four classes of cyclins in higher eukaryotes Cyclin D G1-Cdk Commit cells to DNA replication Cyclin E G1/S-Cdk Initiation of DNA synthesis Cyclin A S-Cdk Initiation of DNA synthesis Cyclin B M-Cdk Promote mitotic events 9/10/2008 PMCB Lecture 9: G. Peter 11

12 Post Translational Control of CDK Activity: Activation Activation Cyclin binding Partial activation Phosphorylation by Cdk-activating kinase (CAK) Fully activated 9/10/2008 PMCB Lecture 9: G. Peter 12

13 Control of CDK Activity: Deactivation Deactivation Phosphorylation by Wee1 Kinase Reversed by cdc25 phosphatase Cdk Inhibitor Binding Reversible Cyclin Degradation by SCF or APC Ubiquitylating enzyme 9/10/2008 PMCB Lecture 9: G. Peter 13

14 9/10/2008 PMCB Lecture 9: G. Peter 14

15 Cell Cycle in Budding Yeast Cdc28 is the only cdk G1-cdk 1 accumulates during G1 but is inactive due to the inhibition by Sic1 binding (CKI) G1-cdk 1activity stimulates synthesis of G1/S-cyclins and G1/S-cdk 1complexes which initiate the events that commit the cell to S phase, stimulating S-cyclin genes, and initiating DNA replication M-cyclins synthesized and activate M-cdk, this commits cells to mitosis and coordinates, nuclear division events Cycle ends with degradation of M-cyclins 9/10/2008 PMCB Lecture 9: G. Peter 15 Biochem. Molec Bio. of Plants, 2000

16 Transcriptional Control of S Phase Induction: The Rb Brake E2F binds to elements in the promoters of many genes that code for proteins required for S phase entry Retinoblastoma (Rb) is an inhibitor of E2F Active G1-cdk phosphorylates Rb and promotes disassociation from E2F to activate S phase gene expression 9/10/2008 PMCB Lecture 9: G. Peter 16 Biochem. Molec Bio. of Plants, 2000

17 Control of DNA Synthesis by S-CDKs Initiation of DNA replication Cdc6 binds to ORC and recruits Mcm proteins to form the pre-rc Essential for initiation but not for ongoing DNA synthesis S-Cdk together with a 2 nd kinase triggers DNA replication Prevention of rereplication S-cdk phosphorylates cdc6 after DNA replication begins which releases it from pre-rc and targets it for degradation S-cdk phosphorylates Mcm subunits which promote export from nucleus (M-cdk also) S-cdk activity stays high through G2 and early mitosis 9/10/2008 PMCB Lecture 9: G. Peter 17

18 Activation of Licensed Origins CDK and Cdc7-Dbf4 kinases are required for activation and work at different stages of the initiation reaction Active CDK phosphorylates cdc18, orc subunits and MCM proteins Cdc7 interacts with Dbf4 to form an active kinase that phosphorylates MCM protein subunits 2,3,4 and 6 Dbf4 levels fluctuate in the cell cycle 9/10/2008 PMCB Lecture 9: G. Peter 18

19 Summary Protein kinase regulated feedback loops Transcriptional control Post translational control Allosteric Phosphorylation Dephosphorylation Protein binding Inhibitors Activators Proteolysis 9/10/2008 PMCB Lecture 9: G. Peter 19

20 Seminal Papers for the Cell Cycle Control Hartwell, L., Culotti, J. and Reid, B. Genetic Control of the Cell Division Cycle in Yeast, I Detection of Mutants (1970) PNAS Hartwell, L., Culott, J., Pringle, J. and Reid, B. Genetic Control of Cell Division Cycle in Yeast (1974), Science Nurse P., Thuriaux P. Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe (1980) Genetics 96(3): /10/2008 PMCB Lecture 9: G. Peter 20

21 Budding Yeast Cell Cycle: Control of G1 Progression Upon exiting M phase G1-cdk accumulates, but needs to be off for growth and extracellular signals to induce the next cell cycle Hct1, a close relative of cdc20, binds APC to keep M-cyclin level low in G1 Hct1-APC activity is high late in mitosis Sic1 is a CKI for M-cdk Hct1-APC, Sic1, and reduced transcription of cyclins act together to ensure early G1 has virtually no cdk activity 9/10/2008 PMCB Lecture 9: G. Peter 21