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Characterization of Budding Yeast Orc6 : a Dimerization Domain Is Characterization of the role of Orc6 in the cell cycle of the budding yeast Saccharomyces cerevisiae by Jeffrey W. Semple A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Biology Waterloo, Ontario, Canada, 2006 © Jeffrey W. Semple 2006 I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required finalrevisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6- eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence. iii Acknowledgements I just want to take this opportunity to thank those that have helped me with this project. First, I would like to thank Dr. Duncker for finding space for me in his lab. I know that this project hasn’t been easy on either of us, but I appreciate all your help in getting it done. As well, I would like to thank Dr. Heikkila for agreeing to co-supervise me and my committee members Dr. Charles and Dr. Dixon, and substitute Christine Dupont, for their time and effort. There are a number of people with whom I have worked with over the past few years that were integral parts of this project. First of all, I would like to thank Angela Varrin, who was always there to help, even when she wasn’t asked. More than simply offering hands-on assistance, she gave me the emotional support required to step into the lab day after day. To Lance “Romance” Da-Silva, I want to thank you for helping with the Orc6 project on-and-off for the past four years, and being a good friend. Also, to the many other faces in the lab that contributed to this work, either through helping with experiments or just putting a smile on my face. I want to thank Kris Rogers, Matt Ramer, Hyder Al-Attar, Jen Sweny, Ajai Prasad, Dave Schnurr, Jessica Steinmoeller, Julie Turnbull and Lutz Kummer. The fact that these individuals are only mentioned briefly is not an indication of the extent of the help they provided. There are also a few individuals outside the laboratory that had a direct impact on my project. Particularly, Eric Jervis for helping develop techniques to effectively visualize my “very small” fluorescent yeast and offering expert advice about imaging, and science in general. To Tania Roberts, Cheryl Smith and Grant Brown from the University of Toronto for helping with FACS analysis and being available for questions. As well, my iv thanks go out to Gwenn Versini and Philippe Pasero from CNRS (Montpellier, France) for being very hospitable when I invaded their lab, helping with all my DNA combing experiments and being there to offer advice regarding my experiments. To Tony Hazbun from the University of Washington and the Yeast Resource Center for all his help with the two-hybrid library screen. Also, I would like to acknowledge Susan Gasser from University of Geneva, for sending strains and antibodies. There are those within the Biology Department at the University of Waterloo that supported me time and time again, even if it was just stopping me in the hall to say hi. I want to thank Steve Wiseman, Thomas D. Singer (from Optometry), Amelie Gravel, Julie Gauley, Mark Lampi, Alison McGuire, Neel Aluru, Darah Christie, Asha Jacob, Brendan McConkey, Mongo Marsden, Bernie Glick, Dragana Miskovic, and Lynn Hoyles. This is not a complete of the people in the department important to me, as there are many others over the past four and a half years that made an impact on my life and project, but I need to put a stop to this list of names somewhere and I apologize for not giving them the credit you deserve. Also, I want to thank Catherine Taylor from Dr. John Thompson’s laboratory for some human cDNA. Finally, I would like to thank my loved ones for their ongoing support. I want to thank my friends for being simply being my friends, I know it isn’t always easy when they don’t see me often and when they do I tend to be a bit stressed. But they were always there to let me drop science for a little while and pick up a beer bottle, which always helped. Particularly, I would like to thank my brother Joe, for being there for support and pulling me away from the lab and onto the golf course when needed. To the first Dr. J. Semple (now known as “C”), it was always important to me to know that you were just down v there hall, and there if I ever needed to take a break. I want to thank you for the free lunches, your car when needed, and yes, your financial support. To my mom, who never really knew what I was doing in the lab, but was there to help regardless, thanks for the everything you did to make my life easier. Finally, I want to thank my girlfriend Shannon. I often wonder how I was able to attract the interest of anyone while I stressed, depressed and frustrated over the past four years, least of all someone as wonderful as you. But you saw through the dark cloud over my head, and your friendship, patience and understanding was, and is, very much appreciated. vi Table of Contents Abstract........................................................................................................... iii Acknowledements.......................................................................................... iv List of Tables .................................................................................................. xi List of Figures ................................................................................................ xii Acronyms........................................................................................................ xv CHAPTER I: Literature Review and Research Objectives .......................... 1 Working with Yeast........................................................................................... 2 A Model organism.......................................................................................... 2 Establishing synchronous cultures of yeast................................................... 4 Alpha factor................................................................................................. 5 Hydroxyurea (HU)....................................................................................... 6 Nocodazole................................................................................................. 7 S. cerevisiae genetics.................................................................................... 8 The Budding Yeast Cell Cycle.......................................................................... 10 G1 phase....................................................................................................... 12 S phase and DNA replication......................................................................... 13 G2 phase....................................................................................................... 17 Mitosis and cytokinesis.................................................................................. 18 Regulation of the cell cycle............................................................................ 20 Initiation of DNA Replication............................................................................. 22 Origins of DNA replication ............................................................................. 22 Timing of origin activation.............................................................................. 25 The pre-replicative complex........................................................................... 26 The origin recognition complex (ORC)........................................................ 29 Cdc6/18 ...................................................................................................... 34 Cdt1 ............................................................................................................ 37 Minichromosome maintenance proteins ....................................................
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