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Regulation of Cdc18 and Cdt1 Restricts S Phase to Once Per Cell

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Regulation of Cdc18 and Cdt1 Restricts S Phase to Once Per Cell Regulation of Cdc18 and Cdt1 Restricts S phase to Once Per Ceii Cycie in Fission Yeast Thesis presented for the degree of Doctor of Philosophy University of London by Stephanie Kim Yanow Cell Cycle Laboratory Imperial Cancer Research Fund 44 Lincoln’s Inn Fields, London Supervisor; Dr. Paul Nurse May, 2001 ProQuest Number: U642466 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642466 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract In all eukaryotes, it is essential that cells maintain a strict control over S phase to ensure that DNA replication occurs once, and only once, per cell division cycle. To achieve this, cells must promote DNA replication during the G1 phase and then repress DNA replication during the G2 phase. In the fission yeast Schizosaccharomyces pombe, two proteins required for the initiation of DNA replication, Cdcl8 and Cdtl, may be important for this control over DNA replication. CdcI8 and Cdtl are both upregulated in G1 and have an essential function in initiation, and are then efficiently downregulated in G2. In this thesis, I have tested whether the downregulation of these initiation factors is important to repress DNA synthesis in G2. I have found that the accumulation of high levels of Cdcl8 are sufficient to induce origin firing in G2 cells and drive re-replication in the absence of mitosis. This re-replication is potentiated by the co-expression of Cdtl, which may be mediated by the stabilisation of Cdcl8 on chromatin by Cdtl. Biochemical characterisation of these re-initiation events revealed that certain aspects of this re-replication are similar to those observed in wild type S phase cells, such as the requirement for the MCMs and the cyclin-dependent kinase Cdc2. However, these cells fail to undergo complete and efficient replication of the chromosomes suggesting that Cdcl8 and Cdtl can override the normal controls that block initiation, but this mechanism does not fully mimic initiation in a G1 cell. The findings presented in this thesis have identified and characterised multiple, overlapping mechanisms that downregulate Cdcl8 and Cdtl in G2. If these mechanisms are disrupted and Cdel8 and Cdtl are allowed to accumulate in G2, replication competence can be re-established and cells re-replicate. Therefore, Cdcl8 and Cdtl form the core of the control which licenses chromosomes for replication. Acknowledgements I’d like to begin by thanking my supervisor, Paul Nurse, for giving me a chance in the first place. I thank him for his support and enthusiasm, and for playing the optimist in the face of my cynicism. It was an invaluable privilege to have Paul as a mentor, one which I will always appreciate and draw upon for inspiration. I would like to say a special thanks to all the members of the Cell Cycle lab, past and present, for their endless help, for their friendship, and for making London home. In particular. I’d like to thank Zoi Lygerou for guiding me through the minefield of biochemistry, my neighbour José Ayté for always knowing the answers and for his patience in explaining them, thanks to Buzz Baum who taught me everything I know about yeast although I hadn’t realised it, and to Jacky Hayles for teaching me everything I know about yeast the second time around. Thank you to Anabelle Decottignies for her friendship and for the Belgian treats that have sustained me through the writing of this thesis. Thank you to Emma Greenwood for being a friend in and outside the lab, and for interesting discussions on the perplexities of CdcI8. I am also grateful to my dear friends from home, Maija Harju, Rachel Somerfield, and Rosalyn Trigger, for indulging me in my rants and raves, for uplifting emails and welcomed diversions from the frustrations of science. Most especially, I would like to thank my parents, Nycole and Joel Yanow, and my brother Stuart for their love, encouragement and interest, and for always standing by me from across the pond. Finally, I would like to thank Wayne Sossin for introducing me to the wonders of lab life all those years ago, and for rescuing me from the cold room on my first day. This PhD was funded by a scholarship from the Natural Sciences and Engineering Research Council of Canada, an Athlone-Vanier Fellowship from the British Council, an Overseas Research Studentship from University College London, by a grant to Paul Nurse from the Association for International Cancer Research, and by the Imperial Cancer Research Fund. This thesis is dedicated to my husband Bradley, for every moment shared and those that lie ahead. Table of Contents Abstract............................................................................................................................ 2 Acknowledgements...........................................................................................................3 Table of Contents............................................................................................................. 5 List of Figures and Tables..................................................................................................8 List of Abbreviations:....................................................................................................... 10 Publications Arising From This Thesis..............................................................................12 Notes on Collaborative Work............................................................................................12 Chapter 1 : An Overview of the Cell Cycle ............................................................... 14 1.1 General overview of the mitotic cell cycle ....................................................................15 1.2 The core cell cycle machinery .................................................................................... 17 1.2.1 Identification o fC d........................................................................................... c2 17 1.2.2 The cyciins.......................................................................................................... 19 1.2.3 Regulation of CDKs: phosphorylation...................................................................22 1.2.4 Regulation of CDKs: cyclin proteolysis................................................................. 25 1.2.5 Regulation of CDKs: CDK inhibitors......................................................................27 1.2.6 The Cdc2 cycie....................................................................................................29 1.3 Regulation of S phase onset ......................................................................................31 1.3.1 The repiicator sequence...................................................................................... 31 1.3.2 The repiication initiator: ORC...............................................................................34 1.3.3 Assembly of the pre-repllcative complex.............................................................. 36 1.3.4 Formation of the pre-initiation complex: .....................................................Cdc45 39 1.3.5 Activation of the pre-initiation complex: S-CDKs and Dbf4/Cdc7............................40 1.4 Mechanisms that restrict S phase to once per cell cycle ..............................................43 1.5 Specific aims .............................................................................................................49 Chapter 2: CdcIS Expression in G2 Cells Induces DNA Synthesis............................. 55 Chapter Summary ...........................................................................................................56 2.1 Introduction................................................................................................................57 2.2 Results ......................................................................................................................58 2.2.1 Cdc18 and Cdtl are downregulated in G2.................................................... cells 58 2.2.2 Cdc18 induces DNA synthesis in G2 cells.............................................................59 2.2.3 Replication origins re-fire illegitimately in G2 cells overexpressing.............. Cdc1861 2.2.4 Association of initiation factors with chromatin during re-replication........... from64 G2 2.2.5 Cdc21 is genetically required for re-repiication............................................ in G2 67 2.3 Discussion................................................................................................................ 68 Chapter 3: Cdt1 Potentiates the Re-replication Inducedby O d d 8 in G 2 ...................... 87 Chapter Summary ...........................................................................................................88 3.1 Introduction............................................................................................................... 89 3.2 Results......................................................................................................................90
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