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Functional and Structural Characterization of the Essential AAA+ Atpases Rvb1 and Rvb2 of S.Cerevisiae

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Functional and Structural Characterization of the Essential AAA+ Atpases Rvb1 and Rvb2 of S.Cerevisiae Functional and Structural Characterization of the essential AAA+ ATPases Rvb1 and Rvb2 of S.cerevisiae by Jennifer Kai Wai Huen A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Jennifer Kai Wai Huen (2014) Functional and Structural Characterization of Rvb1 and Rvb2 of S.cerevisiae Doctor of Philosophy, 2014 Jennifer Kai Wai Huen Department of Biochemistry, University of Toronto Abstract Rvb1 and Rvb2 are essential proteins of the AAA+ superfamily of ATPases associated with a diversity of cellular activities. The Rvbs are components of several chromatin remodeling complexes and the R2TP complex, which is involved in the assembly of small nucleolar ribonucleoprotein complex, RNA polymerase II, Telomerase complex, and Phosphatidyl inositol 3' kinase-related kinases. As such, the Rvbs play an integral role in regulating gene expression, cell signaling, cell growth, and the DNA damage response. Their involvement in these processes is conserved from yeast to human and aberrations in Rvb regulation have been linked to a variety of carcinomas. Because Rvbs are essential in a host of cell processes, defining their molecular and biological functions will aid in future research and application. This work provides a structural, functional, and behavioral characterization of Rvb1 and Rvb2 of S .cerevisiae. Rvb1 and Rvb2 together were demonstrated to form a heterohexameric ring complex. Electron microscopy analysis of the endogenous Rvb1/Rvb2 complex showed similar hexameric rings as those observed for the recombinant Rvb1/Rvb2 complex suggesting that this complex is physiologically relevant. The Rvb1/Rvb2 complex exhibited synergistically enhanced ATP-hydrolysis and DNA unwinding activities in comparison to either subunit alone. The Walker B motif involved in ATP-hydrolysis was found to be essential in both subunits for cell viability and for ATPase activity. Synthetic genetic analysis of RVB1 and RVB2 genes with non-essential genes in S. cerevisiae revealed that both RVBs share a high percentage of genetic ii interactions, indicating that they are both involved in the same cellular pathways. In addition, Rvb1 and Rvb2 are highly co-localized in the nucleus during normal cell growth and under various stress conditions suggesting that their function in the cell resides predominantly in the nucleus and may involve the Rvb1/Rvb2 complex. This work will lend general knowledge towards furthering our understanding of these important proteins and provide a foundation for studies of the human Rvbs in cancer therapies. iii Acknowledgements To the person whom I owe the most gratitude, and whose constant support and encouragement, patience and understanding has been the foundation of all my life pursuits. This thesis is dedicated to my mom. Thanks to my family, especially my sister for her optimism and encouragement, and my dad for letting me do more studying! A special thanks to Eric Cheung, my go-to guy, and his family. I give much thanks to my supervisor (Walid A. Houry) for the chance to work on this project, and for his mentorship throughout these years. His inquisitive mind and enthusiasm for science continues to influence my outlook on the world and beyond. Thanks also to my supervisory committee (Alan Davidson and Craig Smibert) and collaborators (Joaquin Ortega, Kevin Cheung, Ahyoung Park, Yuliya Gordiyenko, Philip Wong, Alex Bezginov, Bryan-Joseph San Luis, Michael Costanzo) for their unique perspectives and contributions to different aspects of this project. Also thanks to my summer and project students for their assistance and dedication (Hussein Zeineddine, Lucie Dupin, Tania Remy). To all the Houry lab: thank you for many years of companionship complete with laughter and tears, food, games, sillyness, and just the right dose of wacky and weird (Frank Zhao, Nardin Nano, Kai Liu, Deji Ologbenla, Hannah Zhao, André Pow, Francisca Ugwu, Majida El Bakkouri). A special mention to Anna Gribun, Yoshito Kakihara, Keith Wong, Taras Makhnevych, Rongmin Zhao, and Guillaume Desjardins for their guidance and wisdom. Thanks also to my dear friends Angela Yu and Usheer Kanjee for helping me maintain balance and sanity. And lots of thanks to Elisa Leung for keeping all our heads above water. Last but definitely not least, thanks to everyone in the Biochemistry Department and other fantastic people throughout the years: Jay Yang, Mod Rujiviphat, Odile Lagacé, Myrna Cohen- Doyle, Kohila Mahadevan, Anthony and Jing Arnoldo, Chris Helsen, Jennifer Tsai, Josephine Yau, Shirley and George King. iv Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements....................................................................................................................... iv Table of Contents ........................................................................................................................... v List of Tables ................................................................................................................................ ix List of Figures ................................................................................................................................ x Abbreviations ............................................................................................................................... xii Published literature not presented in this thesis .......................................................................... xiv Chapter 1. General Introduction ................................................................................................ 1 1.1 Rvb family of AAA+ proteins: conservation of Rvbs across eukaryotes and archaea ... 1 1.2 Structure and oligomerization states of Rvbs in S. cerevisiae and H. sapiens ............... 4 1.3 ATPase and DNA unwinding activity of Rvb1/Rvb2 ................................................... 10 1.4 The physiological function of the Rvbs: components of various macromolecular complexes ................................................................................................................................ 12 1.4.1 The chromatin remodeling complexes: Ino80, Swr1, Tip60, and BAP ................. 12 1.4.2 R2TP in snoRNP assembly .................................................................................... 20 1.4.3 R2TP in the assembly and stabilization of PIKKs ................................................ 21 1.4.4 R2TP in RNAP II assembly ................................................................................... 22 1.4.5 R2TP in TERT assembly ....................................................................................... 24 1.4.6 Rvbs in association with other cellular factors ...................................................... 25 1.4.7 Mechanism of function of R2TP ........................................................................... 26 1.5 Implications of Rvb1 and Rvb2 in development .......................................................... 28 1.6 Implication of Rvb1 and Rvb2 in disease ..................................................................... 30 1.7 Thesis rationale ............................................................................................................. 37 Chapter 2. Yeast Rvb1 and Rvb2 are ATP-Dependent DNA Helicases that Form a Heterohexameric Complex .......................................................................................................... 39 2.1 Abstract ......................................................................................................................... 40 2.2 Introduction ................................................................................................................... 41 v 2.3 Results ........................................................................................................................... 44 2.3.1 The Rvb1 and Rvb2 proteins form a tight heterohexameric complex ................... 44 2.3.2 The Rvb1/2 complex forms a 6-fold symmetrical single-ring particle.................. 46 2.3.3 Rvb1, Rvb2, and Rvb1/2 are active ATPases ........................................................ 49 2.3.4 Observation of the nucleotide-dependent conformational changes in the Rvb1/2 complex by electron microscopy .......................................................................................... 52 2.3.5 Rvb1, Rvb2, and Rvb1/2 are active helicases ....................................................... 54 2.3.6 Purified endogenous Rvb1/Rvb2 complexes are single hexameric rings of similar structure to the in-vitro-assembled complexes with untagged Rvb proteins ....................... 56 2.4 Discussion ..................................................................................................................... 60 2.4.1 The ATPase and helicase activities of the Rvbs .................................................... 60 2.4.2 The oligomeric state of the Rvb1/2 complex ......................................................... 62 2.4.3 Structure of Rvb1/Rvb2 from immunoprecipitated R2TP..................................... 64 2.5 Materials and Methods .................................................................................................. 64 2.5.1 Recombinant protein expression, purification, and manipulation ......................... 64 2.5.2 Size-exclusion chromatography
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