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Copyright by Young-Sam Lee 2010 Copyright by Young-Sam Lee 2010 The Dissertation Committee for Young-Sam Lee Certifies that this is the approved version of the following dissertation: Structural and Functional Studies of the Human Mitochondrial DNA Polymerase Committee: Whitney Yin, Supervisor Ian Molineux Kenneth Johnson Tanya Paull Jon Robertus Structural and Functional Studies of the Human Mitochondrial DNA Polymerase by Young-Sam Lee, B.S, M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2010 Dedication For my wife, In-Sook Jung. Acknowledgements I would like to appreciate Dr. Whitney Yin for giving me chance to working in her lab and mentoring me through my graduate program. Not only the scientific insights, also the warmness that she gave me and my family encouraged me to pursue my Ph. D. degree in the foreign country. I also would like to thank “a guru of molecular biology” Dr. Ian Molineux and “a guru of enzyme kinetics” Dr. Kenneth Johnson. Without their critical advice, I would not be accomplished my publication. I hope to be a respectable expert in my research field like them. I also should remember friendship and generosity given by many current and former Yin lab members: Hey-Ryung Chang, Qingchao “Eric” Meng, Xu Yang, Jeff Knight, Dr. Michio Matsunaga, Dr. He “River” Quan, Taewung Lee, Xin “Ella” Wang, Jamila Momand, and Max Shay. Most of all, I really appreciate my parents for their endless love and support, and my wife, In-Sook Jung, and my son, Jason Seung-Hyeon Lee who always stand by me with patients during my graduate carrier. I will continue to try my best to make them to be proud of me. v Structural and Functional Studies of the Human Mitochondrial DNA Polymerase Publication No._____________ Young-Sam Lee, Ph.D. The University of Texas at Austin, 2010 Supervisor: Y. Whitney Yin The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the vi structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic- phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. vii Table of Contents List of Figures ........................................................................................................ xi List of Tables ....................................................................................................... xiii Chapter 1: Introduction ............................................................................................1 Mitochondrial DNA polymerase (Pol γ) .........................................................1 Enzyme activity of Pol γ ........................................................................1 Accessory subunit Pol γB as a processivity factor for DNA replication3 Pol γ-mutation related mitochondrial disease .................................................4 Mitochondrial toxicity by nucleoside analogue reverse transcriptase inhibitors ................................................................................................................6 Replication Mechanism of human mitochondrial DNA .................................7 Significance of studies in the dissertation .......................................................9 Chapter 2: Cloning, over-expression and purification of the human Pol γ subunits10 Cloning human Pol γ subunits ......................................................................10 Generating a recombinant baculovirus for Pol γA expression in insect cell system. .................................................................................................12 Overexpression of human Pol γA in Sf9 insect cells ....................................12 Human Pol γB overexpression in bacteria ....................................................14 Purification of Pol γ subunits ........................................................................14 Discussion .....................................................................................................17 Initial efforts to express active Pol γA in bacteria ...............................17 Optimizing overexpression and purification procedure for Pol γA expressed from insect cells .........................................................21 Chapter 3: Crystal structure of the human Pol γ holoenzyme and its functional implications ...................................................................................................26 Introduction ...................................................................................................26 Materials and Methods ..................................................................................27 Construction of Pol γA and Pol γB. .....................................................27 Polymerization assay ...........................................................................28 viii Limited proteolysis ..............................................................................28 Crystallography ....................................................................................29 Results and Discussion .................................................................................30 Structure of the catalytic subunit .........................................................30 Holoenzyme formation and subunit interface ......................................35 Processivity of the holoenzyme ...........................................................41 Distinct mode of substrate binding ......................................................48 Pol γ mutations and human diseases ....................................................49 Structural dissimilarities between human Pol γ and HIV reverse transcriptase provides exploitable space for drug design ...........54 Chapter 4: Dissecting the processivity function of the dimeric human Pol γB .....57 Introduction ...................................................................................................57 Materials and Methods ..................................................................................58 Cloning, expression and protein purification .......................................58 Analytical ultracentrifugation ..............................................................59 Steady-state Polymerization assay .......................................................60 Pre-steady state kinetics .......................................................................60 Analytical gel filtration ........................................................................61 Results ...........................................................................................................62 Construction and Preparation of Pol γB variants .................................62 Oligomerization of Pol γB variants ......................................................64 Effects of Pol γB dimerization on processive DNA synthesis .............67 Pre-steady State Kinetics analysis of Pol γB variants ..........................69 Effect of Pol γA on Pol γB dimerization ..............................................72 DNA-dependent subunit interaction ....................................................74
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