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.
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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 genotypicphenotypic 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.
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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
Discussion.....................................................................................................77
Contribution factors to processivity.....................................................77 Distinct roles of each Pol γB monomer in processivity.......................78 Pol γA promotes Pol γB dimerization..................................................80
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Chapter 5: Characterization of R232 mutant of human mitochondrial DNA polymerase: The residue linked to mitochondrial disease controls balance between polymerization and proofreading activities....................................84
Introduction...................................................................................................84 Materials and Methods..................................................................................86
Cloning, expression, and protein purification......................................86 Polymerization kinetics assays ............................................................87 Pre-steady state exonuclease assay......................................................87
Results...........................................................................................................87
Pol γA variants construction and mutation ..........................................87 Interaction between Pol γA R232 variants and the accessory subunit.88 DNA synthesis activity by Pol γA R232 variants................................90 Effect of Pol γA R232 mutation on processivity .................................94 Effects of the R232 mutation on exonuclease activity.........................97 Mismatch removal by Pol γA and holoenzyme .................................100 Hydrolysis of a correct base-pair.......................................................101
Discussion...................................................................................................104
Substitutions of Pol γA R232 alter both polymerase and exonuclease activites of holoenzyme ............................................................105
Pol γA R232 senses the conformation of primer-template for selective exo activity.......................................................................................106
R232 and primer strand transfer ........................................................107 Pol γA R232 substitution and human diseases...................................110
Summary..............................................................................................................112 References............................................................................................................115 Vita .....................................................................................................................121
x
List of Figures
Figure 2.1 Scheme of cloning human Pol γA gene into pBacPAK9 shuttle vector for baculovirus generation...................................................................................................... 11
Figure 2.2 Evaluation of plaque-purified baculovirus for Pol γ overexpression in insect cells.. ................................................................................................................................. 13
Figure 2.3 Overexpression of human Pol γ subunits ....................................................... 14 Figure 2.4 Purification of human Pol γ subunits.............................................................. 17 Figure 2.5 Attempt to obtain soluble Pol γA in bacteria.................................................. 18 Figure 2.6 In-gel activity assay to detect polymerization activity of Pol γA................... 19 Figure 2.7 Attempt to isolate functional domains of Pol γA after trypsin digestion ....... 20
Figure 3.1 Polymerization activities of Pol γA (ΔN-exo-) and Pol γB (ΔI4) ................... 31 Figure 3.2 Structure of human Pol γ holoenzyme. (A) Structure of Pol γA .................... 34 Figure 3.3 Formation of Pol γ holoenzyme analyzed by gel filtration chromatography . 35 Figure 3.4 The major Pol γ subunit interfaces ................................................................. 39 Figure 3.5 DNA activity assay of Pol γA-L-helix mutant ............................................... 40 Figure 3.6 Superposition of Pol γA and T7 DNAP shows their similarity in the active sites. .................................................................................................................................. 41
Figure 3.7 Comparison of a modeled Pol γ-DNA complex with that of the T7 DNAP- DNA complex................................................................................................................... 44
Figure 3.8 Mapping Pol γB deletion mutants on the Pol γ holoenzyme structure........... 45 Figure 3.9 Structure of Pol γB-ΔI4 dimer........................................................................ 46 Figure 3.10 Pol γA charge distribution............................................................................ 47 Figure 3.11 Pol γA mutational analysis ........................................................................... 52 Figure 3.12 Structural differences between human Pol γ and HIV reverse transcriptase 56
Figure 4.1 Structural and bioinformatic basis for construction of Pol γB variants.......... 63 Figure 4.2 Variant Pol γB oligomeric states .................................................................... 66 Figure 4.3 Steady-state DNA polymerization assays ...................................................... 68 Figure 4.4 Time-dependent product formation in pre-steady-state assays ...................... 71
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Figure 4.5 Effects of Pol γA and DNA on Pol γB dimerization ...................................... 75 Figure 4.6 Subunit interaction between Pol γA and ΔI4-D129K..................................... 76
Figure 5.1 Interaction between Pol γA R232- Pol γB E394 in the Pol γ holoenzyme ......... 89 Figure 5.2 Steady-state DNA synthesis activities of Pol γA R232 mutants ...................... 92 Figure 5.3 DNA polymerase activities of holoenzymes containing Pol γB E394 variants 93 Figure 5.4 Pre-steady-state single nucleotide incorporation............................................ 96 Figure 5.5 Exonuclease activities of Pol γA R232 variants............................................. 98 Figure 5.6 Exonuclease analyses ................................................................................... 103 Figure 5.7 The signaling pathway in the modeled Pol γ holoenzyme-DNA complex... 110
xii
List of Tables
Table 3.1 Statistics of data analysis and structural refinement......................................... 32 Table 3.2 Classification of disease-related Pol γA substitutions ...................................... 53
Table 4.1 Dissociation constants measured by analytical ultracentrifugation.................. 66 Table 4.2 Pre-steady state kinetic parameters for Pol γB variants.................................... 71
Table 5.1 Primer set for the mutagenesis of human Pol γ subunits .................................. 86 Table 5.2 Polymerization activities of Pol γA mutants..................................................... 96 Table 5.3 Global fitting analysis for exonuclease activity of Pol γA variants.................. 99 Table 5.4 Exonuclease activity of Pol γA variants ......................................................... 103 Table 5.5 Selective exonuclease activities of wild-type and mutant enzymes ............... 103
xiii
Chapter 1: Introduction
MITOCHONDRIAL DNA POLYMERASE (POL γ)
DNA replication is an essential process to maintain genetic integrity. In eukaryotic cells, mitochondria are unique subcellular organelles which contain their own DNA (mitochondrial DNA, mtDNA) distinct from nuclear chromosomal DNA. Mitochondria are the power houses of the cells, generating ATP through the process of oxidative phosphorylation (OXPHOS). Because essential protein components for the OXPHOS are encoded in mitochondrial as well as in chromosomal genome, integrity of mtDNA is critical for the cellular energy supply and activities.
Replication of mtDNA is performed by several nuclear-encoded proteins that form mitochondrial nucleoid (1). Among them, Pol γ is the sole protein serving DNA polymerization for mtDNA replication. Animal Pol γ consists of two subunits, a 140-kDa catalytic subunit, Pol γA, and a 55-kDa accessory subunit, Pol γB.
Enzyme activity of Pol γ
Pol γ is a high-fidelity DNA polymerase with 5’ Æ 3’ polymerization activity for
DNA synthesis as well as 3’ Æ 5’ exonucleolytic activity for proofreading; catalytic residues for the reactions are in the catalytic subunit Pol γA. Although Pol γA by itself exhibits a salt-sensitive polymerization activity, forming a complex with Pol γB creates a Pol γ holoenzyme which is more salt resistance in a range of 0~200 mM salt concentration. (2). The rate of polymerization also increases from 9 sec-1 to 45 sec-1 upon Pol γA complex with the Pol γB (3). Interestingly, the enzyme exhibits a broad substrate spectrum using templates that are not only DNA, but also RNA. Therefore it is thought to contain reverse transcriptase activity (4). Although the specificity constant for incorporation of dNTPs in RNA-dependent DNA polymerization by the Pol γ is 25~100
1fold lower than the value in DNA-dependent DNA polymerization (5), the reverse transcriptase activity is perhaps important to Pol γ, because it is known that mtDNA contains ribonucleotides (6), estimated to be at least 30 per a molecule of rat liver mtDNA (7).
The exonuclease activity of Pol γ contributes to the fidelity in mtDNA replication by hydrolysis of misincorporated nucleotides during DNA synthesis. The conserved negatively charged residues in the catalytic core of the exo domain (D198 and E200) are known to be essential for the activity (8). By kinetic measurements, exonuclease deficient (E200A)-Pol γ makes one error per 280,000 base pairs copied (8), and the exonuclease activity increases the fidelity by about 200-fold (9).
Pol γ also has 5’-deoxyribose phosphate (dRP) lyase activity that is important for
DNA repair (10). The enzyme removes 5’-dRP groups generated by the activity of glycosylases and abasic endonucleases during base excision repair process. However, because the enzyme-dRP intermediate is very stable, Pol γ appears to have a much slower turnover rate than Pol β that is specialized for the repair process, which suggests that Pol γ might serve the repair function in low frequency (10).
Some enzymes that lack of 3’Æ 5’ exonuclease activity (exo-) such as Taq polymerase or 3’Æ 5’ exo- E. coli DNA polymerase I can extend the 3’-terminus of blunt-ended DNA by one nucleotide, using exclusively dAMP, in a non-template dependent manner (11). Exo- Pol γ variants also appear to non-template nucleotide addition, especially with dATP, although the reaction efficiency is low (Y.-S. Lee and Y.W. Yin, unpublished observation). The terminal transferase activity of Pol γ might not be relevant in vivo because of the exonuclease activity of the wild-type enzyme, but it might be interesting to study Pol γ in relation to other types of template-independent
2nucleotide addition such as incorporating nucleotides opposite abasic sites done by many translesion synthesis polymerases (12).
Based on comparison of amino acid sequences with other DNA polymerases, Pol γA contains common motifs of a polymerase (pol) and an exonuclease (exo) domain; these are shared among DNA Pol I family (or family A polymerase), including E. coli DNA Pol I Klenow fragment and T7 DNA polymerase (gp5) (13). N-terminal exo domain and C-terminal pol domain are linked by a spacer which has no sequence homology with other members of the family (14). Biochemical studies showed that deletion or mutation of some residues in the spacer affect polymerization and/or Pol γB interaction (15). In T7 DNA polymerase, this region is involved in interacting with its processivity factor, thioredoxin, and expanding interaction area with DNA (16).
Accessory subunit Pol γB as a processivity factor for DNA replication
Many replicative DNA polymerases require a processivity factor for efficient
DNA replication because their catalytic subunits alone can incorporate only a few nucleotides per DNA binding event. This is likely due to their rapid dissociation from template DNA (17). Although Pol γA displays high (~100 nt synthesis per binding) processivity relative to other catalytic subunits of DNA polymerases (18), Pol γB serves as a processivity factor for efficient mtDNA replication with Pol γA.
The processivity mechanism by Pol γB is distinct from the other processivity factors. Mammalian Pol γB is structurally homologous to class II aminoacyl tRNA synthetases (19,20), and has no resemblance to other processivity factors showing a toroidal shape like PCNA for eukaryotic DNA polymerase δ and ε (21) or small globular such as thioredoxin for T7 DNA polymerase (16). Even functionally, Pol γB not only increases the DNA binding affinity with the replicase (2), but also enhances the
