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Structure Function Analysis of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles in Kinetoplast Maintenance

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Structure Function Analysis of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles in Kinetoplast Maintenance University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations Dissertations and Theses July 2020 STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE Jonathan Miller University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Molecular Biology Commons Recommended Citation Miller, Jonathan, "STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE" (2020). Doctoral Dissertations. 1938. https://doi.org/10.7275/17466180 https://scholarworks.umass.edu/dissertations_2/1938 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE A Dissertation Presented by JONATHAN C. MILLER Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2020 Department of Microbiology © Copyright by Jonathan C. Miller 2020 All Rights Reserved ii STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE A Dissertation Presented By Jonathan C. Miller Approved as to style and content by: Michele M. Klingbeil, Chair Steven J. Sandler, Member Yasu S. Morita, Member Peter Chien, Member James F. Holden, Department Head Department of Microbiology iii DEDICATION To my father Michael who gave me the resources and support to find my own path to success, To my brother Shawn for being the voice of reason and always protecting me, To my mother Kimlien for her tireless support to provide a better future for her children, To Cindy who without I wouldn’t be the person I am today. Thank you. iv ACKNOWLEDGEMENTS First and foremost I must thank my supervisor and mentor Dr. Michele Klingbeil, who enrolled me in biochemistry bootcamp many summers ago and taught me how to become an independent thinker at the chalkboard and a leader in laboratory. I thank my committee members Drs. Steven Sandler, Yasu Morita, and Peter Chien for their intellectual guidance and support throughout this process. Thank you to all the teaching faculty I had the privilege of teaching under and alongside as they gave me a love and respect for teaching and inspiring students. To the many members of the Klingbeil lab over the years: Jeny CA, Mike B, Rebecca G, Garvin D, Stephanie D, Yadi B, Maria CR, Jirka T, David A, Sam K, Dan B, Elsie H, Paris J, Jon K, Parker C, James H, Thanh B, Loan V, and Matt F. I have learned so much from you and I hope I could teach you something helpful in your respective journeys as well. Thank you to my UMass graduate school family spanning over a half dozen departments across the Amherst campus, especially my dear friends Jen H and Michael U who I wouldn’t have made it through my first graduate school years without their unconditional support. I would also be remiss to not mention Emily R, Julia P, Gina C, and Bia D for all our adventures across the state and abroad! Also, thank you to the house members for adopting me during my surgery recovery process. v Thank you to my family and friends for a what was quite a number of years of supporting me. Thank you to Cindy for completing my graduate eXperience with much-needed love and care. vi ABSTRACT STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE MAY 2020 JONATHAN C MILLER B.S., UNIVERSITY OF CONNECTICUT, STORRS PH.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Michele M. Klingbeil Kinetoplastid organisms including medically relevant species like Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy number and sequence classes among different species, kDNA is most often found as a network of thousands of DNA molecules catenated together. This unique biological property of disease- causing organisms has been a subject of study as a potential drug target since it is essential for parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and related diseases in other mammals and is fatal if left untreated. Recent advances in drug development have produced an oral medication to treat most forms of the disease, hopefully alleviating the medical burden of the organism and enabling us to appreciate and study its fascinating mitochondrial biology. Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. vii In Chapter 2, we show that POLIC localization changes throughout kDNA S phase and in order to look for potential interactions with kDNA segregation machinery, we utilize structured illumination microscopy to follow localization dynamics with a TAC marker, TAC102. Additionally, we show that kDNA replication proteins POLIB and POLID do not affect TAC102 localization further reinforcing them as strictly kDNA replication proteins. In Chapter 3, we use an RNAi- complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation. viii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................................... v ABSTRACT ................................................................................................................................. vii LIST OF TABLES .......................................................................................................................... xii LIST OF FIGURES ....................................................................................................................... xiii CHAPTER 1. LITERATURE REVIEW AND STUDY BACKGROUND .................................................................... 1 1.1 The global health impact of Trypanosoma brucei ................................................... 1 1.2 Infection and immunity of Trypanosoma brucei ..................................................... 3 1.3 Structure, sequence, and function of kDNA ........................................................... 5 1.4 Spatiotemporal and structural aspects of kDNA network replication ..................... 9 1.5 Mechanistic insight into kDNA replication ............................................................ 10 1.6 Segregation of kDNA networks ............................................................................ 15 1.7 Goal of this study ................................................................................................. 17 1.8 Significance and contribution of this study ........................................................... 17 1.9 Bibliography ......................................................................................................... 23 2. INVESTIGATION OF POLIC CELL CYCLE LOCALIZATION IN RELATION TO TRIPARTITE ATTACHMENT MARKER, TAC102 ............................................................................................... 34 2.1 Abstract ............................................................................................................... 34 2.2 Introduction ......................................................................................................... 34 2.2.1 Widefield fluorescence microscopy and its limitations .................................. 34 2.2.2 Principles of super-resolution microscopy ..................................................... 36 2.2.3 Principles of Structured Illumination Microscopy .......................................... 38 2.2.3 Cell cycle dependent localization of kDNA-associated proteins ..................... 39 2.3 Materials and Methods ........................................................................................ 40 2.3.1 DNA constructs. ............................................................................................ 40 2.3.2 Trypanosome cell culture and transfection ................................................... 41 2.3.3 Widefield Immunofluorescence .................................................................... 41 2.3.4 Structured Illumination Microscopy .............................................................. 42 2.3.5 Three-dimensional reconstruction and projection ........................................
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