:X. :vlfluzi . :1 I . l a La...” 3 i . a“, 1 n a . “may? . 1...... s -x ”3.315". as}... chi—1mm: awry, 3.1. .0 3. x “V. x ”3%....” . . . :32 thwlxfl r... {.31 , .n. . .3 a. . . Q I I p x! u. 3' .‘xwa‘xn 67.1 u¢s‘,...r.v 5.2.11.1 . . . . . A , p . . _ . . . V ‘ 7.. u), r u p f .5 cm . I I :. 2 i: 31:13:: . 1:: E: u‘ fa . 3:458 1007 LIBRARY R inhrnor ‘3+""‘f\ l IIUIIIHUII ULGLU University This is to certify that the thesis entitled MODULAR ARCHITECTURE AND DYNAMIC OLIGOMERIC STRUCTURE OF THE HUMAN MITOCHONDRIAL REPLICATIVE DNA HELCIASE presented by Tawn Denise Ziebarth has been accepted towards fulfillment of the requirements for the Master of degree in Biochemistry and Molecular Science Biology W! 5. WI.” 0. Major Professor’s Signature WWW t7r'L Zoab Date MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/C|RC/DaleDue.indd-p.1 MODULAR ARCHITECTURE AND DYNAMIC OLIGOMERIC STRUCTURE OF THE HUMAN MITOCHONDRIAL REPLICATIVE DNA HELICASE By Tawn Denise Ziebarth A THESIS Submitted to Michigan State University in partial fitlfillment of the requirements for the degree of MASTERS OF SCIENCE Department of Biochemistry and Molecular Biology 2006 ABSTRACT MODULAR ARCHITECTURE AND DYNAMIC OLIGOMERIC STRUCTURE OF THE HUMAN MITOCHONDRIAL REPLICATIVE DNA HELICASE By Tawn Denise Ziebanh We have purified a recombinant form of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication, and probed its structure. Substantial amino acid sequence and functional similarities of this novel helicase are shared with the bacteriophage T7 primase-helicase, a member of the DnaB-like family of helicases. We show in velocity sedimentation and gel filtration analyses that the mitochondrial helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-terminal sequencing reveals distinct N- and C-terminal polypeptides that represent minimal structural domains. Truncations of the N- and C-termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is functional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and alignments, combined with biochemical data, suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. Physical analysis of the proteolytic products defines the region required to maintain an oligomeric structure to reside within amino acids ~405-590, and DNA binding to occur between motifs II-IV. We have found that tri-nucleotide binds in motif I and stabilizes the enzyme by inducing a conformational change to an oligomer beyond hexamer/heptamer. Our findings place the human mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases. For Brooke, my daughter and my best fiiend. I love you! iii ACKNOWLEDGMENTS The completion of this thesis can be attributed to the constant support of Dr. Laurie Kaguni, my mentor and friend. My initial acceptance by Dr. Kaguni into the BRTP opened a door of interest for me that continued to grow into my graduate degree and beyond and I feel truly fortunate to have studied under her supervision. Her influence and guidance has reshaped my life. I will always be grateful to Dr. Kaguni for taking a chance on such an inexperienced scientist. Wherever my scientific career takes me, I will always credit my future achievements to Dr. Kaguni and I will be forever appreciative for her training and support. Thank you to Carol Farr, without whom I would not have survived my first month in the BRTP program. I am grateful to her for showing me everything, for her expertise and her contributions to this work. You are a great scientist and a good friend. Special thank you to my daughter, Brooke. I realize how time consuming this has been and I want you to know I appreciate your understanding. I am very proud and happy to have you as my daughter. I have every confidence that you will have great accomplishments in your life and I will always be there to help you achieve them. Thank you to my parents for their love and encouragement. Without them this would not have been possible. And thank you for always listening to my work related “dilemmas”. I love you both very much! iv Thank you to my brother, Tim, and his family. The desire to prove him wrong was very motivating and kept me going. Thank you to Robert Montgomery for his patience and support. You are a great partner and confidant. Your positive attitude and giving nature was always reassuring during this very challenging time. Your sense of humor and silliness was my escape and kept me sane. Thank you to my Thesis Committee members: Dr. Zachary Burton and Dr. William Wedemeyer for advice and guidance. Thank you to Dr. Jon M. Kaguni for recommendations pertaining to my research during our joint lab meetings. Thank you to members of both Kaguni labs past and present including: Alec Murillo for always helping me with all my computer “issues”; Yuichi Matsushima, Sam Kim, Sundari Chodavarapu, Magda Felczak and Magdalena Makowska-Grzyska. Thank you God for giving me a second chance. I might not have deserved one but will do everything in my power to fulfill my purpose. TABLE OF CONTENTS Page LIST OF FIGURES .............................................................................. viii LIST OF ABBREVIATIONS ..................................................................... ix CHAPTER I GENERAL INTRODUCTION .................................................................... 1 Mitochondria and Mitochondrial DNA replication .................................... 2 The Mitochondrial Replisome ............................................................ 3 Bacteriophage T7 gene 4 protein 5 CHAPTER II MODULAR ARCHITECTURE OF THE HEXAMERIC HUMAN MITOCHONDRIAL DNA HELICASE ......................................................... 9 Summary ................................................................................... 10 Introduction ................................................................................ 1 1 Experimental Procedures ................................................................ 14 Materials .......................................................................... 14 Methods ........................................................................... 15 Results ...................................................................................... 20 The human mitochondrial DNA helicase has a hexarneric quaternary structure .............................................................. 20 The hexarneric human mitochondrial DNA helicase is a modular protein ............................................................................. 23 Oligomeric properties of the human mitochondrial DNA helicase proteolytic products ............................................................. 33 ATPase activity of the human mitochondrial DNA helicase and its proteolytic products ............................................................. 36 Discussion ................................................................................. 39 CHAPTER HI COFACTOR EFFECTS ON THE SOLUBILITY, QUATERNARY STRUCTURE AND CONFORMATIONAL CHANGES OF THE HUMAN MITOCHONDRIAL DNA HELICASE ..................................................................................... 44 Summary ................................................................................... 45 Introduction ................................................................................ 46 Experimental Procedures ................................................................ 49 Materials .......................................................................... 49 Methods ............................................................................ 49 Results ...................................................................................... 52 vi Page Factors stabilizing the human mitochondrial DNA helicase under physiological ionic conditions .......................................... 52 Oligomeric states of the human mitochondrial DNA helicase in the presence of cofactors ................................................... 55 Trypsin digestion of the human mitochondrial DNA helicase reveals conformational changes upon cofactor and substrate binding ........................................................................... 58 Discussion ................................................................................. 61 CHAPTER IV SUMMARY AND FUTURE PERSPECTIVES .............................................. 70 Summary ................................................................................... 71 Future Perspectives ....................................................................... 72 LIST OF PUBLICATIONS- Tawn D. Ziebarth ............................................. 75 BIBLIOGRAPHY ................................................................................. 76 vii LIST OF FIGURES Page Figure l — Glycerol gradient sedimentation and gel filtration of the human mitochondrial DNA helicase ............................................. 22 Figure 2 - Trypsin and multiple protease digestion of human mitochondrial DNA helicase ............................................................................... 26 Figure 3A — Schematic map of human mitochondrial DNA helicase proteolytic products ................................................................ 28 Figure 3B - Secondary-structure prediction of human mitochondrial DNA helicase ............................................. 30 Figure 4 — Trypsin time course digestion of human mitochondrial DNA helicase .............................................. 32 Figure 5 — Gel filtration of human mitochondrial DNA helicase proteolytic products ................................................................ 35 Figure 6 — ATPase activity of human mitochondrial DNA helicase and truncated forms ..................................................................... 38 Figure 7 — Factors stabilizing the human mitochondrial DNA helicase under physiological conditions ..................................................... 54 Figure 8 — Oligomeric states of the human mitochondrial DNA helicase in the presence of cofactors ......................................................... 57 Figure 9 — Trypsin digestion of the human mitochondrial DNA helicase reveals conformational changes upon cofactor and substrate binding ................. 60 Figure 10 — Structure ofthe thCM double hexarner 64 Figure 11 — DNA binding loop homology between T7 gp4 and the human mtDNA helicase ......................................................... 68 viii ATP ATPase ATPYS DNA dTTP ds E. coli His M g2+ mtDNA thCM PAGE PBS PMSF p01 7 LIST OF ABBREVIATIONS Angstrom amino acid ATPases associated with various cellular activities adenosine diphosphate adenosine triphosphate adenosine triphosphatase adenosine 5’-['y-thio] triphosphate Celsius deoxyribonucleic acid deoxythyrnidine triphosphate double-stranded Escherichia coli hexa-histidine magnesium mitochondrial DNA Methanobacterium thermoautotrophicum MCM nucleoside triphosphate polyacrylamide gel electrophoresis phosphate-buffered saline phenylmethylsulfonyl fluoride DNA polymerase gamma ribonucleic acid ix RPD SDS ss SSB STI T7 g4p T4 g4lp ZBD RNA polymerase domain Stoke’s radius sodium dodecyl sulfate single-stranded single-stranded DNA binding protein soybean trypsin inhibitor bacteriophage T7 gene 4 protein bacteriophage T4 gene 41 protein zinc binding domain CHAPTER I GENERAL INTRODUCTION Mitochondria and Mitochondrial DNA Replication It is widely accepted that mitochondria evolved from a bacterial ancestry. About 1.5 million years ago, the earth’s atmosphere became oxygen-rich and it is believed that this is when a eukaryotic cell engulfed an oxygen-metabolizing bacteria as an adaptation to its new environment (1-3). The organisms became symbiotic and the newly acquired bacteria provided an efficient energy source for the eukaryotic cell. Mitochondria are the direct decendents of that symbiotic bacteria. Mitochondria are double-membraned organelles that provide the majority of energy that eukaryotic cells need for basic survival. To produce this energy in the form of ATP, the mitochondria utilize processes within their membrane known as the citric acid acid cycle and oxidative phosphorylation. Also housed within their inner membrane is the electron transport chain that harnesses electron energy to drive the ATP synthesis reaction. The number of mitochondria within each individual cell varies depending on the ever-changing energy requirements of the particular cell. Each mitochondrion contains multiple copies of a 16 kb double-stranded, circular DNA molecule. This DNA encodes l3 polypeptides required for oxidative phosphorylation in addition to 24 tRNAs and 2 rRNAs used in the translation of its proteins (4). To date, all of the proteins known to be involved in the replication of mitochondrial DNA (mtDNA) are encoded by nuclear genes. The process regulating their expression or mitochondrial transport has yet to be explained. The mechanism of replication is also not entirely understood and continues to be an issue of controversy. Currently, two models exist. The displacement loop model states that replication is unidirectional and asymmetric with leading and lagging strand synthesis occurring continuously (5). The second model, known as the strand-coupled model, defines replication in a symmetric, discontinuous manner as evident by 2D gel electrophoresis data (6,7). Strong experimental evidence supports both models and it is possible that the mitochondrion’s mechanism is mandated by external physiological factors. Of the two, the long-standing displacement loop model is more highly accepted as the predominant means of mtDNA replication. This mechanism requires three main functions for the creation of new complementary strands: unwinding, priming and polymerization. These three fimctions are carried out by the mitochondrial replisome. The known proteins within the replisome include a DNA polymerase gamma (pol y), a single-stranded DNA binding (SSB) protein and a DNA helicase. The enzyme possessing the primase activity has yet to be discovered. The Mitochondrial Replisome DNA polymerases use a two-metal ion mechanism to catalyze the phosphoryl transfer reaction for the synthesis of new complementary strands during replication (8). The mitochondrial replication system operates with a sole DNA polymerase, pol 7, that contains 5’-3’ DNA polymerase activity as well as a 3’-5’ exonuclease proofi'eading function (9). It is a highly processive, two-subunit enzyme made up of a catalytic alpha subunit and an accessory beta subunit (10,11). The alpha subunit, pol y—alpha, consists of two functional domains separated by a spacer region (12). Its exonuclease activity resides in the I, II, and III conserved sequence motifs located in the N-terminal region of the protein. The C-terminal region contains motifs A, B, and C that catalyze its DNA polymerase activity. The spacer region has four conserved sequence blocks recently demonstrated to fitnction in enhancing catalytic activity and enzyme processivity (12). The beta subunit, pol y-beta, is made up of three structural domains and its function is to enhance primer binding and increase pol y processivity and activity (13). A mitochondrial SSB protein participates in multiple cellular activities including DNA repair, recombination and replication. During replication it binds to and stabilizes the lagging strand while preventing reannealing of the separated double helix, and it stimulates the DNA polymerase activity (14-16). It is a homotetrameric protein whose DNA binding properties closely resemble those of Escherichia coli (E. coli) SSB. (17). They share a 36% sequence similarity within their corresponding N-terminus but the mitochondrial SSB is completely devoid of the C-terminal third of E. coli SSB, an area believed to be involved in protein-protein interactions (18). The crystal structure of the human mitochondrial SSB revealed that single-stranded DNA (ssDNA) wraps around the tetrameric protein and that disordered regions exist at both the N—terminus and C- terminus (19). Primases are enzymes that catalyze the synthesis of oligoribonucleotides to serve as primers for elongation by the DNA polymerase during DNA replication. They initiate leading-strand synthesis once and lagging-strand synthesis multiple times during the course of replication. Although essential to both current models of mitochondrial replication, the protein responsible for primase function has not been identified and only partial purifications of such an activity have been reported (20,21). In some systems, such as bacteriophage T7, this activity can be found in a bifunctional enzyme that serves as both the primase and helicase during replication (22). Helicases are a class of enzymes that use the energy of nucleotide hydrolysis to translocate along double-stranded DNA (dsDNA) producing a single-stranded substrate for the DNA polymerase (23,24). They can be classified into five superfarnilies based on the specific features shared among the associated members of that family, and common structural attributes contribute to the appropriate family designation of these proteins. The family consisting of the replicative helicases, also known as the DnaB-like family or SF-4, contains proteins that assemble into hexamers to unwind DNA in a 5'-3' direction (25,26). These enzymes comprise of five conserved motifs including the classic Walker A and Walker B motifs shown to be directly involved in the nucleotide hydrolysis reaction (27). These fimctional motifs are found in a conserved alpha/beta domain referred to as the RecA-like core. Typically, helicases are modular proteins containing this core region plus additional structural domains that specify mechanistic features including substrate specificity and polarity of movement. As a result of the high conservation of the motifs, potential helicase candidates can easily be identified through sequence alignments as demonstrated by the human mtDNA helicase (28). The evolutionary hypothesis that mitochondria evolved within eukaryotic cells from ancient bacteria is not only evident in its functional aspect but also with respect to its DNA replication. Comparisons of mitochondrial replication with numerous prokaryotic replicating systems such as, bacteriophage T7, bacteriophage T4 and E. coli reveal significant similarities among the protein participants and in particular, those within the bacteriophage T7 system. Bacteriophage T7 gene 4 protein In bacteriophage T7, DNA replication occurs by a bidirectional, symmetric and semi-discontinuous mechanism (29). The replication fork comprises only 4 proteins. The T7 gene 2.5 protein serves as the SSB protein, the DNA polymerase function resides in the T7 gene 5 protein along with E. coli thioredoxin acting as its processivity factor, and a biftmctional enzyme known as the T7 gene 4 protein (T7 gp4) functions as both the helicase and primase during this essential process (30-32). The T7 gp4 is made up of 566 amino acids and is a member of the DnaB-like replicative helicases. It assembles into a hexarneric ring producing a central channel for the threading of ssDNA as it translocates in 5'-3' direction (33). It has a conserved alpha/beta core domain found in all helicases along with an additional all beta domain (25). Each protomer has a modular architecture consisting of three distinct structural domains separated by proteolytically-sensitive sites (34,35). The N-terminus functions as the primase and contains two subdomains. The N-terminal zinc-binding domain (ZBD) recognizes and binds the DNA template and subsequently delivers a primed-template to the DNA polymerase active site (36). This domain is separated by a nonstructured flexible tether from the RNA polymerase domain (RPD) that is responsible for oligoribonucleotide synthesis in a template-dependent manner (37). A linker region, found to be important in the oligomerization of the protein, separates the N—terminal primase from the C-terminal helicase domain (38). Limited proteolysis of the T7 gp4 produces a protein termed 4D that consists of residues 241-566 (34). The crystal structure of this active, hexarneric helicase-only domain was solved (39). The structure illustrates that the interface between two neighboring subunits contains the classic Walker A and Walker B motifs found in all helicases. These motifs contain conserved residues directly involved in nucleotide binding and hydrolysis and therefore make up the nucleotide binding pocket. Loops that project into the central channel are involved in contacting ssDNA during translocation. The structure also revealed that the subunits within the hexamer have an asymmetric orientation. It was shown that two of the monomers have symmetry (A subunits), its neighboring subunits are rotated 15 degrees from this symmetry (B subunits) and the remaining two subunits are rotated 30 degrees in the opposite direction to compensate for any torsional strain produced in the oligomer (C subunits). This asymmetry serves a functional purpose by creating non-identical nucleotide binding sites from identical subunits. This produces a negative cooperativity during nucleotide binding by correctly positioning a critical arginine residue, known as the arginine finger (R522), to ligate the nucleotide gamma phosphate in the A and B subunits but not in the C subunits. During nucleotide hydrolysis, water causes the displacement of the gamma phosphate on the nucleoside triphosphate molecule, subsequently releasing either inorganic phosphate, nucleotide diphosphate or both. Because the gamma phosphate is bound to Arg522, its liberation causes a repositioning of the arginine residue and triggers a conformational change in the enzyme to open up and expose a DNA binding site in the central channel. The release of product from one subunit binding site causes the hydrolysis of nucleotide bound at another subunit site and illustrates the previously mentioned cooperativity (40) The exact mechanism for coupling the energy released from the hydrolysis reaction to the translocation along DNA is uncertain but crystal structures have helped postulate a plausible model known as the binding change mechanism (39). In this model, two high-occupancy sites bind dTTP, while two low occupancy sites bind dTDP +Pi, and the remaining two sites are empty due to the position of the arginine finger. This indicates that binding and hydrolysis are sequential due to the asymmetric nature of the hexamer. Therefore, when the subunits rotate due to nucleotide binding and hydrolysis, it causes the ssDNA to be passed from one subunit to another, and the loops move in paddle-like motions that spirals the ssDNA through the cavity in the process (39,40). The primase of the T7 g4p not only provides the primer for the DNA polymerase, but it is also necessary for the utilization of that primer by delivering the primed DNA template to the DNA polymerase (36,41). This serves to both stabilize the primer- template complex and secure the primer in the DNA polymerase active site. The N- terminal primase crystal structure, containing residues 1-255, was solved in the presence of two magnesium Mgz+) ions bound to the active site (41). This structure revealed that the primase domain is monomeric and contains a flexible linker between the ZBD and the RPD. The ZBD is a four-stranded antiparallel beta sheet flanked by a C-terminal alpha helix. The zinc binding site is partially exposed on the surface of the protein that faces away from the active site. Therefore, the zinc does not interact with nucleotide or DNA and probably plays a role in fold stability. A 2-D NMR investigation of the T7 gp4 primase region found that, in the absence of DNA, the ZBD and RPD have little interaction. But, in the presence of DNA primed-template, the domains do interact (41). CHAPTER II MODULAR ARCHITECTURE OF THE HEXAMERIC HUMAN MITOCHONDRIAL DNA HELICASE Summary We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. Substantial amino acid sequence and functional similarities of this novel helicase are shared with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-tenninal sequencing reveals distinct N- and C-terminal polypeptides that represent minimal structural domains. Physical analysis of the proteolytic products defines the region required to maintain oligomeric structure to reside within amino acids ~405-590. Truncations of the N- and C-termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is fimctional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and secondary structural alignments combined with biochemical data suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. The hexarneric conformation and modular architecture revealed in our study document that the mitochondrial DNA helicase and bacteriophage T7 primase-helicase share physical features. Our findings place the mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases. 10 Introduction A nuclear gene known as Twinkle encodes the human mtDNA helicase (42). A mitochondrial link to human disease first allowed its identification in individuals afflicted with autosomal dominant progressive external opthalmoplegia, a disease of external eye muscle paralysis associated with multiple mtDNA deletions. That Twinkle might be the mitochondrial replicative helicase was suggested by its apparent amino acid (a) sequence homology with T7 gp4, a bi-functional primase-helicase (28). Biochemical characterization of a recombinant human mtDNA helicase demonstrated its NTPase and DNA unwinding capabilities (43). UTP was found to be the preferred nucleotide substrate, and a 5 '-ss stretch of DNA and a short 3 '-ssDNA tail are required for translocation and duplex unwinding in a 5 '-3 ' direction. Physiological studies reveal that the enzyme is essential for mtDNA maintenance and regulation of mtDNA copy number (28,42). All helicases contain a conserved alpha-beta domain, known as the 'RecA-like' core. This core region houses the functional Walker A and Walker B motifs that contain the residues responsible for nucleotide hydrolysis (25,27). In addition, these enzymes comprise additional distinct domains illustrating an overall modular architecture. The accessory domains contribute to mechanistic specificity by providing directionality or substrate specificity, and include the broad functions of the helicases, which are all members of the larger family of AAA+ ATPases (25). The T7 gp4 is a well-characterized, bifunctional enzyme that serves as a model for studies of novel replicative helicases. It functions as a heterohexarner with protomers that comprise five conserved helicase motifs, placing it in the E. coli DnaB-like family of replicative helicases (25,26). T7 gp4 shares this association with other hexarneric ll helicases such as bacteriophage T4 gene 41 protein (T4 gp41) and the RepA protein encoded by plasmid RSF 1010 (44-47). Electron microscopic studies of these multimers reveal a subunit ring assembly that produces a central channel for the threading of DNA (26,48,49). Early physical studies of T7 gp4 and DnaB protein identified common proteolytically—sensitive regions (34,35,50). The hill-length T7 gp4 consists of three distinct structural and functional domains that are separated by flexible linkers. Its N- terrninal domain contains two subdomains, ZBD and a RPD (41,51). The ZBD recognizes and binds to a DNA priming site (5'-GTC-3') and subsequently delivers the primed-template to the DNA polymerase active site (41). The RPD synthesizes an oligoribonucleotide primer that is extended by the T7 DNA polymerase, T7 gene 5 protein (36,52). The C-terminal helicase domain of T7 gp4 contains the TTPase activity and is solely responsible for unwinding dsDNA by displacing the complementary strand (34,36,39). The N- and C-terminal domains are separated by a linker that is important for oligomerization and also mediates conformational changes (38,53). E. coli DnaB protein functions as a DNA helicase during bacterial DNA replication and interacts with its corresponding primase, E. coli DnaG protein (46,54). The DnaB protein monomer contains two structural domains separated by a hinge region (50). Its N-terminal domain dimerizes under certain conditions to regulate the quaternary structural changes that the protein undergoes, and is essential for helicase activity (49,55,56). The hinge participates in the protein-protein interactions with DnaG protein, and residues in the C-terminal domain comprise its DNA binding, oligomerization and ATPase activities (42,50,55). Variations among the DnaB-like enzymes include the regions responsible for 12 oligomerization. The majority of subunit interactions in T7 gp4 involve helix A (aa 272- 281), that lies C-terminal to the linker of one monomer, interacting with helices D1-D3 (aa 345-388), a region in between motif IA and motif H on a neighboring monomer. Additional interaction contributions come from specific residues in the linker region (3 8,53,57). In the RSFl 010-encoded RepA protein, the extreme N-terrninus, residues 2- 18 of one monomer, interact with residues 108—1 14 of the adjacent monomer (48). In contrast, the N-termini of both T7 gp4 and DnaB protein are readily cleaved by limited proteolysis with retention of oligomeric structure (34,50). To date, high-resolution crystal structures are available for several members of the DnaB-like family of replicative helicases, including T7 gp4 and RepA protein (48,58). The T7 gp4 lacking its N-tenninal ZBD shows a structural asymmetry that suggests a mechanism of sequential hydrolysis to explain its translocation along DNA (39). RepA protein, on the other hand, shows complete symmetry among its subunits (48). Findings similar to those for RepA protein were reported in electron microscopic studies of the E. coli DnaB protein under specific conditions (49). The differences in subunit orientation among the helicases argue against a common mechanism of nucleotide hydrolysis leading to translocation (25,57,59,60). Instead, such mechanistic diversity may be relevant for cellular versatility as demonstrated in other hexarneric ATPases (61-63). To evaluate the structural features of the human mtDNA helicase, we pursued a combined approach of limited proteolysis and hydrodynamic methods. We demonstrate the modular architecture of the hexameric enzyme. We evaluate our findings in comparison with those for other fimctionally-homologous proteins, placing the human mtDNA helicase in the DnaB-like family of replicative helicases. 13 Experimental Procedures Materials Enzymes and Protein Standards- All proteases, soybean trypsin inhibitor (STI), bovine serum albumin, bovine liver catalase, rabbit muscle L—lactate dehydrogenase, and yeast alcohol dehydrogenase were purchased from Sigma. Color markers for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) were also purchased from Sigma. [a-32P]dATP was purchased from MP Biosciences. E. coli DnaB protein was a gift of Dr. Jon Kaguni of this department. Hexa-histidine (His) -tagged monoclonal antibody was purchased from Becton Dickinson Biosciences. Polyethyleneimine chromatography paper impregnated with fluorescent indicator was purchased from Brinkrnan Instruments, Inc. Chemicals- Amphotericin, penicillin-G, streptomyocin and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma. PMSF was prepared as a 0.1M stock solution in isopropyl alcohol and stored at -20° C. Sodium metabisulfite (J. T. Baker Chemical Co.) was prepared as a 1 M stock solution at pH 7.5 and stored at -20° C. 1,4- Dithio-DL-threitol was purchased from Research Organics. Leupeptin was purchased from the Peptide Institute, Minoh-Shi, Japan, and was prepared as a 1mg/ml stock solution in 50 mM Tris-HCl, pH 8.0, 2 mM EDTA and stored at 4° C. SDS for gel electrophoresis was from Pierce. Nickel-nitrilotriacetic acid resin was purchased from Qiagen. Other Materials- Spodopterafiugrperda cells were the gift of Dr. Suzanne Thiem. TC-IOO insect cell culture medium and fetal bovine serum were from Life Technologies, 14 Inc. Insect cell transfection buffer and Grace’s medium were purchased from PharMingen. Baculoviruses encoding N- and C-tenninally His-tagged human mtDNA helicase (a 43-684) were a gift of the F alkenberg lab at the Karolinska Institute. Baculoviral plasmid DNA encoding N-terminally His-tagged human mtDNA helicase P66 (a 43-633) and N-terminally His-tagged human mtDNA helicase P57 (aa 146-633) were constructed by and purchased from ATG Laboratories, Inc. A Superdex 200 HR 10/30 column was purchased from Amersham Biosciences. Methods Construction of Recombinant Baculoviruses P66 and P5 7-Linearized wild-type baculovirus DNA (BaculoGold, 0.5 ug; PharMingen) and purified transfer plasmid DNAs (ATG Laboratories) encoding P66 or PS7 human mtDNA helicase were co-transfected in transfection buffer containing 25 mM HEPES, pH 7.1, 125 mM CaClz, 140 mM NaCl for 4 h at 27° C following the manufacturer’s recommendations. Recombinant viruses were amplified in Spodopterafrugiperda cells to titers of approximately 2 X 108 plaque forming units/ml. Protein Overexpression and Purification of Human mtDNA Helicase- Spodoptera frugiperda cells were grown in TC-lOO insect culture cell medium containing 10% fetal bovine serum and infected with recombinant viruses at a multiplicity of infection of 5 at 27 ° C. Cells were harvested at 72 h post infection by centrifirgation and washed with an equal volume of cold phosphate-buffered saline, recentrifirged, frozen in liquid nitrogen and stored at -80° C. The frozen cell pellets were thawed on ice, and all further steps were performed at 0-4° C. Cells were suspended in 1/45 volume of original cell culture in 25 15 mM Tris-HCl, pH 8.0, 1 mM PMSF, 10 mM sodium metabisulfite, 2 jig/ml leupeptin, 10 mM 2-mercaptoethanol. Cells were lysed in a Dounce homogenizer using 20 strokes of the tight pestle. The homogenate was adjusted to a final salt concentration of 1 M NaCl followed by centrifugation. The soluble extract (Fr 1) was diluted with an equal volume of 50 mM Tris-HCl, pH 8.0, 0.6 M NaCl, 10% glycerol and loaded onto a nickel- nitrilotriacetic acid agarose coltunn (2.5 ml resin/liter cells) equilibrated with buffer containing 50 mM Tris-HCl, pH 8.0, 0.6 M NaCl, 10% glycerol, 10 mM imidazole, 1 mM PMSF, 10 mM sodium metabisulfite, 2 [Lg/ml leupeptin, 10 mM 2-mercaptoethanol. The column was washed with the equilibration buffer containing 0.6 M NaCl and 10 mM imidazole, and the bound protein was eluted with buffers containing 25 mM, 250 mM and 500 mM imidazole. Recombinant proteins eluted at 250 mM imidazole; fi'actions were analyzed by SDS-PAGE and silver staining, and fractions were pooled accordingly as Fr 11. Fr II was diluted to an ionic equivalent of 150 mM NaCl and loaded onto a heparin sepharose column (2.7 mg/ml resin) equilibrated with buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5 mM EDTA, 1 mM PMSF, 10 mM sodium metabisulfite, 2 11.ng leupeptin, 10 mM 2-mercaptoethanol. The column was washed in the equilibration buffer containing 200 mM NaCl and the bound protein was eluted with buffers containing 0.6 and 1 M NaCl. Recombinant proteins eluted at 350- 700 mM NaCl; fractions were analyzed by SDS-PAGE followed by silver staining, and pooled accordingly as Fr 111. Fr III was loaded onto 12-30% glycerol gradients in 35 mM Tris-HCl, pH 7.5, 330 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM sodium metabisulfite, 2 jig/ml leupeptin, 10 mM 2-mercaptoethanol. Gradients were centrifirged at 37,000 rpm for 30 h. Fractions were analyzed by SDS-PAGE and silver staining, and pooled accordingly as Fr IV. Fr IV was frozen in liquid nitrogen and stored in aliquots at 16 -80° C. Analytical Glycerol Gradient Sedimentation of Human mtDNA Helicase-N- and C-His-tagged human mtDNA helicase (Fr IV) was layered onto preformed 12-30% glycerol gradients containing 35 mM Tris-HCl, pH 7.5, 330 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM sodium metabisulfite, 2 jig/ml leupeptin, 10 mM 2-mercaptoethanol. Protein standards of known S values run in parallel were: jack bean urease, 18.6 S; bovine liver catalase, 11.3 S; rabbit muscle L-lactate dehydrogenase, 7.3 S. Centrifugation was at 37,000 rpm for 30 h at 4° C. Fractions were analyzed by SDS- PAGE and silver staining. The data were plotted as S value versus fraction number to obtain a sedimentation coefficient for the human mtDNA helicase. Gel F iltration- C-terrninal His-tagged human mtDNA helicase (200 pg) was chromatographed on a Superdex 200 HR 10/30 column (10 x 300-310 mm, Amersham Biosciences) equilibrated with buffer containing 35 mM Tris-HCl, pH 7.5, 330 mM NaCl, 8% glycerol, 1 mM EDTA at a flow rate of 0.25 ml/min at 4° C. The column was calibrated with chymotrypsinogen A (R5 = 28 A), hen egg ovalbumin (R5 = 30 A), yeast alcohol dehydrogenase (R5 = 46 A), bovine liver catalase (R5 = 52 A) and bovine thyroid thyroglobulin (R3 = 85 A), where R5 is the Stoke’s radius. Fractions (0.25 ml) were collected and aliquots were examined by SDS-PAGE and silver staining to confirm ultraviolet trace recordings. Limited Proteolysis-N- and C-terrninally His-tagged human mtDNA helicase (2 pg) was proteolyzed in 50 mM Tris-HCl, pH 8.0, 50% glycerol, 4 mM MgC12, 2 mM 2- 17 mercaptoethanol for 10 min at 20° C. The trypsin titration contained a trypsin concentration range of 0.05 pg - 10.0 pg. The multiple protease digestion used optimal helicase: protease ratios (w/w) as follows: trypsin 1:1, chyrnotrypsin 1:0.35, thermolysin 1:1 (for 40 min), subtilisin 1:0.005, papain 1:0.18. The trypsin time course digestion used a trypsinzhelicase ratio of 1 :1. At various time points indicated in the legend of Figure 4, 30 pl aliquots were removed from the digested sample. All trypsin digestions were stopped by the addition of STI at a STI: trypsin ratio of 2: 1. All of the other protease digestions were stopped by addition of 10 mM sodium bisulfite, 2 ug/ml leupeptin, and 1% SDS. Digestion products were separated using SDS-PAGE, and analyzed by silver staining and/or immunoblotting. N—terminal Sequencing- Following trypsin digestions designed to optimize production of individual fragments, proteolytic products were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane in CAPS buffer at a constant current of 150 mAmp for 16 h. The membranes were stained with 0.05% R250 Coomassie Blue followed by destaining in 40% methanol. The proteolytic fragments were visualized, cut from the membrane and submitted for N-terminal sequencing analysis by standard Edman chemistry. Preparation of Proteolytic Products for Gel Filtration Analysis- To produce T57/T50, C-terrninally His-tagged mtDNA helicase was proteolyzed at a helicase:trypsin ratio of 1:2 for 10 min at 20° C, and the digestion was stopped with STI. To produce T34/T 28, C-terminally His-tagged mtDNA helicase was proteolyzed at a helicase:trypsin ratio of 1:0.25 for 55 min at 20° C and stopped with STI. T57/T50 and T34/T28 were 18 chromatographed on the Superdex 200 HR 10/30 column and analyzed as for the full- length protein. AT Pose Assay- Reaction mixtures (20 pl) contained 20 mM Tris-HCl, pH 7.5, 4 mM MgC12, 0.1 mg/ml bovine serum albumin, 10 % glycerol, 0.5 mM ATP, 10 mM dithiothreitol, 4 pCi of [a-32P]dATP, 100 pM DNase I-activated calf thymus DNA, and N- and C-terminally His-tagged full-length mtDNA helicase, T66/T 57, T5 7/T 50, P66, P5 7 and T28. Incubation was for 15 min or 0-40 min time course at 37° C. The reactions were stopped by addition of EDTA to 20 mM and by placing the reaction tubes on ice, and two micoliter aliquots were spotted onto Polygram polyethyleneimine cellulous paper with 0.5 pl of 50 mM ADP/ATP. The Polygram polyethyleneimine cellulous paper was developed in 1 M formic acid, 0.5 M LiCl. The position of both ADP and ATP bands was visualized by ultraviolet light. The corresponding bands were isolated and the radioactivity was measured by liquid scintillation counting. 19 Results The human mtDNA helicase has a hexamcric quaternary structure The human mtDNA helicase was purified previously to near-homogeneity in four chromatographic steps from baculovirus-infected insect cells (43). We developed a streamlined purification strategy using N— and C-terminally His-tagged forms of the protein for our physical studies (see Methods). This procedure yielded 0.7 mg of near- homogeneous enzyme per liter of infected cells. To investigate the oligomeric state of our recombinant human mtDNA helicase, we used hydrodynamic methods to determine its molecular mass. Velocity sedimentation produced a single peak with a sedimentation coefficient of 13.6 S (Fig. 1A). Similarly, gel filtration of the enzyme produced a single peak with a Stokes radius of 80 A (Fig. 1B). Combining the sedimentation coefficient with the Stokes radius yields a native molecular mass of 420,000 daltons. SDS-PAGE of the velocity sedimentation and gel filtration peak fractions reveals a 72,000 dalton monomeric polypeptide. Taken together, our data indicates that the purified, recombinant human mtDNA helicase exists as a hexamer, and is consistent with the prominent oligomeric state of the members of the E. coli DnaB-like family. 20 Fig. 1A Glycerol gradient sedimentation of human mtDNA helicase. Helicase was sedimented in a 12-30% glycerol gradient. Standard protein markers run in parallel gradients were: Jack bean urease (URE, 18.6 S), bovine liver catalase (CAT, 11.3 S), and rabbit muscle lactate dehydrogenase (LDH, 7.3 S). Fig. 1B Gel filtration of human mtDNA helicase. Helicase was chromatographed on a Superdex 200 column. The standard protein markers were: chymotrypsinogen (R, = 28 A), hen egg ovalbumin (R, = 30 A), yeast alcohol dehydrogenase (R, = 46 A), bovine liver catalase (R, = 52 A), and bovine thyroid thyroglobulin (R, = 85 A). 21 > o—S l 0 1i) 2i) 30 Fraction number 201 Band intensity, Arbitrary units I T r r fifir 0 10 20 30 40 Fraction number OJ C I l 1 Fraction number . § 104 Band intensity, Arbitrary units N o l O O I I I 0 10 20 30 40 50 Fraction number Figure 1A. Glycerol gradient sedimentation of human mtDN A helicase Figure 1B. Gel filtration of human mtDNA helicase 22 The hexarneric human mtDNA helicase is a modular protein A modular architecture is a structural characteristic of T7 gp4 and E. coli DnaB (35,50). We investigated whether the human mtDNA helicase comprises distinct structural domains by proteolyzing the enzyme using a titration of trypsin under limiting conditions, and identified six prominent cleavage products of 66 (T66), 57 (T57), 50 (T50), 34 (T34), 30 (T30), and 28 (T28) kDa (Fig. 2A). Furthermore, limited proteolysis with a variety of proteases with diverse cleavage specificities showed that each of the products of trypsin digestion was produced by more than one protease (Fig. 2B). The initial trypsin product, T66, was evident upon silver staining and immunoblot analyses of both N- and C-terrninally His-tagged human mtDNA helicase. However, immunoblots probed with His monoclonal antibody detected only the N-terminally His- tagged T66, illustrating cleavage and a loss of the His-tag from the carboxyl terminus (data not shown). Based on these data, we conclude that T66 derives from cleavage near a residue 640, and represents a human mtDNA helicase lacking its C-tenninus (Fig. 3A). For each of the proteolytic fragments, the position of the C-terminus was estimated from the migration in SDS-PAGE, the cleavage specificity of trypsin and a secondary structural prediction indicating non-structured regions (Fig. 3B). N-terminal sequencing demonstrates that T57 is the product of subsequent cleavage at the N-terminus of T66. It is produced by cleavage at a residue K144, and creates a polypeptide lacking both termini. Furthermore, N-terminal sequencing of the T50, T34 and T28 fragments demonstrates that they all share a common N-terminus with T57. We conclude that there is a progressive digestion of T57 at its C-tenninus until a minimal fragment is produced, T28. T30 is produced from cleavage at a residue R371, and represents a polypeptide spanning aa ~3 72-640. A time course of trypsin digestion demonstrates the stable nature 23 of each fragment, and shows that the two polypeptides T30 and T28 represent minimal N- and C-terrninal structural domains (Fig. 4). 24 Fig. 2A Trypsin titration of human mtDNA helicase. Human mtDNA helicase was proteolyzed with increasing concentrations of trypsin. Samples were analyzed by SDS- PAGE followed by silver staining. Ratios are given as helicase:trypsin. Lane 1, undigested helicase; lane 2-8, 1: 0.02, l: 0.08, l: 0.2, l: 0.3, 1: 0.6, l: 1.3, 1: 2.0. Fig. 28 Multiple proteases produce similar stable fragments of human mtDNA helicase. Human mtDNA helicase was digested with five different proteases, and the products were analyzed by SDS-PAGE followed by silver staining. Ratios are given as helicase:protease. Lane 1, undigested helicase; lane 2, trypsin 1:1; lane 3 chyrnotrypsin 1:0.35; lane 4, thermolysin 1:1; lane 5, subtilisin 120.005; lane 6, papain 1:0.15. 25 . 72 mtDNA helicase — —- .3--- - .4 66 “so _ - J34 _ — 30 . “28 trypsrn— B mtDNA helicase -— ’ Figure 2A. Trypsin titration of human mtDNA helicase Figure 2B. Multiple proteases produce similar fragments of human mtDNA helicase 26 Fig. 3A Schematic map of proteolytic fragments of human mtDNA helicase. 27 N-terminal region linker C-terminal region ( ): =( > 1 I IA II III IV 684 aa mtDNA helicase [ 43 ~640 T66 .9 : T57 I45 ~642 T3 4 I45 ~42 T30 E72 «649 145 ~406 T28 : : Figure 3A. Schematic map of proteolytic fragments of human mtDNA helicase 28 Fig. BB Sequence alignment of human mtDNA helicase and bacteriophage T7 helicase-primase. A secondary structure-based sequence alignment was produced using 3D-PSSM (ww.sbg.bio.ic.ac.gk). 3D-PSSM is a web-based method to determine secondary-structure potential of a query protein on a 3D sequence profile of a known protein. Human mtDNA helicase was used as a probe and T7 gp4 was used as the known protein. Barrels designate alpha helices and arrows designate beta sheets. The 5 conserved amino acid motifs in superfamily IV DNA helicases are indicated by labeled boxes. 29 Hs T7 HS T7 Hs T7 Hs T7 HS T7 HS T7 Hs T7 Hs T7 Hs T7 Hs T7 Hs T7 Hs T7 ‘ -IID dilalfli- 1 MWVLLRSGYPLRILLPLRGEWMGRRGLPRNLAPGPPRRRYRKETLQALDMPVLPVTATEI IIIIIF II‘» n...» 61 RQYLRGHGIPFQDGHSCLRALSPFAESSQLKGQTGVTTSFSLFIDKTTGHFLCMTSLAEG 121 SWEDFQASVEGRGDGAREGFLLSKAPEFEDSEEVRRIWNRAIPLWELPDQEEVQLADTMF 64 ------------------------------------------ MTYNVWNFGESNGRYSAL IEflD- {IIEEEED- Illlib- IIIDr III-I‘D» 181 GLTKVTDDTLKRFSVRYLR-PARSLVFPWFSPGGSGLRGLKLLEAKCQGDGVSYEETTIP 82 TARGISKETCQKAGYWIAKVDGVMYQVADYRDQNGNIVSQKVRDKDKNFK -------- TT III-Id.» Ill-Ilnlqalp- lIlII} fill IIID’ 4IIIIIIIID- IIOt 24o RPSAYHNLFGLPLISRRDAEVVLTSRELDSLALNQSTGLPT ----- LTLPRGTTCLPPAL 134 GSHKSDALFGKHLWNG—GKKIVVTEGEIDMLTVMELQDCKYPVVSLGHGASAAKKTCAAN n—u>- ’- ‘IIIIIIIIIIIIIF dIIIIIIlIIIIIIIIIIIIIIIr 4IIIIIIIIIIII. 295 LPYLEQFRRIVFWLGDDLRSWEAAKLFARKLNPKRCFLVRPGDQQPRPLEALNGGFNLSR 193 YEYFDQFEQIILMFDMDEAGRKAVEEAAQVLPAGKVRVAVLPCKDANECHLNGHDREIME ilfilfilfllllll>> -(IIIEIIIEEIIII¥ Iii} 355 ILRTALPAWHKSIVSFRQLREEVLGELSNVEQAAGLRWSRFPDLNRILKGHRKGELTVFT 253 QVWNAGPWIPDGVVSALSLRERIREHLSS-EESVGLLFSGCTGINDKTLGARGGEVIMVT Motif I m_ mm— A B 1 4IIIIIIIIIIIII¥ IIIID> -CIIIIIIIIIIIF *IIIIIIIIII 415 GPTGSGKFTFISEYALDLCSQ-G TLWGSFEISNVRLARVMLTQFAEGRL ------- ED 312 SGSGMVMSTFVRQQALQWGTAMGKKVGLAMLEE VEETAEDLIGLHNRVRLRQSDSLKRE Motif I Motif IA ‘> , -' III‘> -1I!Iiiiililifl> (I3t4IEZZEL C Di DZ I-D' {IIIIIIIIIIIIP Iii}: 'CID 467 QLD——KYDHWADRF-EDLPLYFMTFHGQQSIRTVIDTMQHAVYVYDICHVIIDNLQFMMG 372 IIENGKFDQWFDELFGNDTFHLYDSFAEAETDRLLAKLAYMRSGLGCDVIILDHISIVVS Moti II EEZEfEEEIZZZZZD' III}: ‘.§L_____;L_JD' Illéi’ D3 3 E 4 -CIIIIIIIIIIIIIIIIIF Ill-II.» {IIIII 524 HEQLSTDRIAA DYIIGVFRKFATDNNCHVTLVI RKEDDDK ----- ELQ-TASIFGSA 432 ASGESDER-KMIDNLMTKLKGFAKSTGVVLVVIC KNPDKGKAHEEGRPVSITDLRGSG Motif III Motif IV -CE!-e ;- ‘ -- 3* ‘1EZZZZZIZEF F 5 G1 IIIflI-Pllllflfib Ill-ll...» III-II‘ID- I‘t 578 KASQEADNVLILQDRKLVTGPGKRYLQVSKNRFDGDVGVF-PLEFNKNSLTFSIPPKNKA 491 ALRQLSDTIIALERNQQGDMPNLVLVRILKCRFTGDTGIAGYMEYNKETGWLEPSSYSG- Mo:if IV 6 7 8 9| G 637 RLKKIKDDTGPVAKKPSSGKKGATTQNSEICSGQAPTPDQPDTSKRSK 684 551 EESHSESTDWSNDTDF -------------------------------- 566 Figure 3B. Secondary-structure prediction of human mtDNA helicase 30 60 120 180 81 239 133 294 192 354 252 414 311 466 371 523 431 577 490 636 550 Fig. 4 Time course of trypsin digestion. Human mtDNA helicase was proteolyzed over a time course of 1.5 h. Samples were extracted at the indicated time points, and analyzed by SDS-PAGE followed by silver staining. Lane 1, undigested helicase; lanes 2-8, 1, 5, 10, 20, 30, 60, and 90 min. 31 mtDNA helicase — id: trypsin - Figure 4. Time course of trypsin digestion 32 Oligomeric properties of the human mtDNA helicase proteolytic products To elucidate the regions responsible for maintenance of the hexarneric form of the enzyme, we examined the oligomeric properties of the proteolytic products, T66/T 57, T57/T 50 and T34/T 28. Samples were subjected to gel filtration under the elution conditions used to obtain the Stokes radius of the intact enzyme. T66/T 57 demonstrated an elution profile similar to the full-length enzyme (Fig. 5A). Using an appropriate trypsin: helicase ratio to ensure complete proteolysis of T66 but optimal production of T57 and T50, we observed a single chromatographic peak in fractions 36-45 (Fig. 5B). SDS-PAGE and silver staining reveal both T57 and T50, consistent with their presence in a hexarneric oligomer. We also used a trypsin: helicase ratio appropriate to ensure maximal production of T28 with minimal retention of the larger proteolytic products. Upon gel filtration of this digest we observed a single chromatographic peak eluting in fractions 65-67, consistent with the presence of T28 eluting as a monomer (Fig. 5C). Therefore, we conclude that the region required for retention of an oligomeric structure resides within aa ~405-590. 33 Fig. 5 Gel filtration of trypsin products T66/T57, T57/T50 and T34/T28. Human mtDNA helicase was subjected to limited proteolysis with trypsin and the samples were fractionated by gel filtration on a FPLC Superdex 200 column. Fractions containing hexarneric (A, B) or monomeric protein (C) were analyzed by SDS-PAGE and silver staining. A) T66/T 57. Lane 1, undigested helicase; lane 2, gel filtration load; lane 3- 7, hexameric peak fractions #3 8-42. B) T57/T50. Lane 1, undigested helicase; lane 2, gel filtration load; [one 3-6, hexarneric peak fractions #39-42. C) T34/T 28. Lane 1, gel filtration load; lanes 2-4, monomeric peak fractions #65-67; lane 5, trypsin. 34 ._.___ _- ___, 1": ._..- .. -T66 — -—- -- -—"-"“ “T57 B 1 2 3 4 5 6 ....- — _. __,.-__, _'r57 ‘— " ”f" "T50 and C 1 2 3 4 5 6 _r34 3": ‘— " ..- ””8 Figure 5. Gel filtration of trypsin products T66/T 57, T57/I50 and T34/1‘ 28 35 ATPase activity of the human mtDNA helicase and its proteolytic products To reveal structure-function relationships in the proteolyzed forms of the human mtDNA helicase, we evaluated their DNA-dependent ATPase activities relative to the full-length enzyme (Fig. 6). First, we investigated whether the tennini contribute to nucleotide hydrolysis by purifying recombinant proteins corresponding to polypeptides T66 and T5 7, which we refer to as P66 and P57, respectively (see Methods). Consistent with our initial gel filtration studies of the proteolytic products, we found the P66 and P57 proteins to be hexamers upon purification (data not shown). Remarkably, the homogeneous P66 that lacks its C-tenninus displays reproducibly increased ATPase activity as compared to the similar activities of full-length and the homogeneous P57, a polypeptide lacking both termini, in two preparations of each protein. These data suggest that the C-terminus of the intact polypeptide is inhibitory to ATPase activity, whereas the N-terminus apparently contributes to the overall catalytic activity of the human mtDNA helicase. In contrast, the heterogeneous oligomer of T57/T50, at ratios of 4:1 and 3:1, exhibits a 3- to 4-fold decrease in activity. Taken together with the data obtained for the homogeneous P5 7, we conclude that the lower activity of the T5 7/T50 complex is due to the lack of critical aa residues in T50. 36 Fig. 6 Time course of ATP hydrolysis by trypsin products. F ull-length human mtDNA helicase (open circles), P66 (closed circles), P57 (open triangles), T57/T 50 (4:1, closed triangles and 3:1, open squares) were assayed for ATPase activity over 0-40 min. 37 ATP hydrolyzed, % N W 4}- UI G \1 e e a: e e o pa ‘13 O IIIIrIITIIIIIIlIIIIIIIIII 0 5 10 15202530 3540 Time, minutes Figure 6. Time course of ATP hydrolysis by trypsin products 38 Discussion The human mtDNA helicase shares significant sequence similarity and functional characteristics with T7 gp4 (28,43). Multiple sequence alignments have demonstrated the conservation of the five highly-conserved helicase motifs, and our secondary structural prediction of the mitochondrial enzyme aligned with the known structure of the bacteriophage enzyme lacking its ZBD using 3D-PSSM suggests additional sequence similarities that likely pertain to structure. We investigated this possibility by examination of the native structure of the recombinant human mtDNA helicase using velocity sedimentation and gel filtration. Spelbrink et al. showed previously that the human mtDNA helicase forms oligomers in vivo (28). Our data demonstrate that the recombinant human mtDNA helicase exists as a hexamer under the ionic conditions of our purification, a common conformation for the active form of replicative helicases (33,44). Our limited proteolysis study demonstrates that the human mtDNA helicase and bacteriophage T7 gp4 share similar structural domains. Remarkably, comparison of our trypsin titration of the human mtDNA helicase with an Endoprotease Glu-C titration of T7 gp4 reveals indistinguishable patterns (34). The two homologous, hexarneric proteins also show comparable progressions of digestion. In both enzymes, the first polypeptide produced is generated from a loss of the C-terminus. Interestingly, a trypsin digestion of the T4 gp41, the replicative helicase in the T4 system, has an initial cleavage in this same region (64). The N-terminal region of T7 gp4 comprises two structural domains, a ZBD and a RPD that are separated by a nonstructured tether (41,51). This tether region is the next site of cleavage and results in a loss of the ZBD. Digestion of the human mtDNA 39 helicase follows this pattern with proteolytic cleavage at an accessible homologous site in its N-terminal region. In contrast, we were unable to demonstrate a N—terminal cleavage product corresponding to the ZBD of T7 gp4 despite several attempts employing different strategies, and thus find no evidence that it exists as a stable product. However, we found that the trypsin-sensitive site at aa R275 in T7 gp4 reported by Washington et al. corresponds to a cleavage occurring at the highly conserved R371 in the human mtDNA helicase. This is consistent with the presence of a region that is structurally-homologous to the linker region of the bacteriophage protein. Our sequence analysis of the proteolytic products of the human mtDNA helicase . also reveals similarities to another member of the hexarneric family of replicative helicases, E. coli DnaB protein. Under limited conditions, a trypsin digestion of DnaB protein produces N- and C-terminal structural domains from a single cleavage occurring in a proteolytically-sensitive region (50). The trypsin digestions in our study also generated N-and C-tenninal polypeptides whose cleavage sites correspond to predicted unstructured regions of the intact protein. Based on the digestion pattern observed with DnaB protein, we would propose that the T30 and T28 polypeptides of human mtDNA helicase result from cleavage of T57 at a position C-terrninal to aa 371. However, it is not clear how this relates to the alternate production of T50 and T34 from T57. Without knowing the precise C-tenninus of T28, we are unable to confirm a hypothesis of alternative proteolytic pathways, and cannot at this time demonstrate the pathway leading to fragment T28. We speculate that multiple proteolytic patterns are indicative of either conformational changes or asymmetry within the hexamer. Regardless, experiments using multiple proteases and time course product stability analyses document that the minimal polypeptides T30 and T28 represent distinct structural domains, and establish 40 the modular architecture of the protein. The majority of the T7 gp4 oligomerization properties are due to interactions between helix A and helices D1-D3 (3 8,53,57). We show that a hexarneric form is still retained in T50 but lost in T28, which exists as a monomer. This suggests that the region containing the residues responsible for maintaining a stable oligomer lies within a 405- 590, and that this region encompasses a homolgous region comprising helices D1-D3 in the bacteriophage protein. Although T28 contains the predicted helix A, it lacks helices D1-D3 and therefore would be incapable of the necessary interactions yielding a stable hexamer. This is consistent with findings reported for the monomeric primase region of bacteriophage T7 gp4 (52). Studies of the bacteriophage T7 and T4 replicative DNA helicases have revealed the importance of their C-termini for protein-protein interactions within the replisome (64,65). In particular, this region in T7 gp4 provides a regulatory role involved in coordinating leading and lagging DNA strand synthesis. However, in neither bacteriophage system has the C-terrninus been shown to affect nucleotide hydrolysis. Interestingly, we show a significant increase in nucleotide hydrolysis for the recombinant P66 protein lacking its C-terminus. Thus, the C-terminus of the full-length enzyme apparently provides a negative regulatory function. In contrast, nucleotide hydrolysis is decreased to full-length enzyme activity in the recombinant P57 protein that also lacks its N-terminus. Our data suggests that the N-terminus contributes to nucleotide hydrolysis in the full-length protein similar to Methanobacterium thermoautrophicum MCM (thCM), an enzyme which often serves as a model of eukaryotic systems (66). In contrast, an E. coli DnaB fragment lacking its N-terminus retains the nucleotide hydrolysis activity of the full-length enzyme (50). In T7 gp4, the N-terminal region 41 contains the ZBD, which contacts DNA and pauses the enzyme during primer synthesis (67). Furthermore, a crystal structure of thCM shows that this region in the archaeal replicative helicase is responsible for mediating dodecamerization via zinc motifs (68). The contribution of the C- terminus to the activity of the mtDNA helicase is novel among the replicative helicases, and the involvement of the N-terminus in this firnction suggests the possibility of cellular functions extending beyond its role in replication. We found that a hexarneric P57 exhibits higher ATPase activity as compared to heterohexarneric T5 7/T50, and note that T50 lacks residues critical to the nucleotide hydrolysis reaction. In particular, we propose that the reduced activity results from the loss of a putative arginine finger: R614 of the human mtDNA helicase likely serves a role analogous to R522 in T7 gp4 (39). The arginine finger, found in all helicases, is responsible for ligating the y-phosphate during the nucleotide hydrolysis reaction. Upon hydrolysis, the release of the y—phosphate displaces the arginine finger, leading to a conformational change and subsequent DNA translocation (39,48,57). The crystal structure of the T7 gp4 demonstrates that within the hexamer subunits A and B are rotated 15° with respect to each other, and the C subunits are rotated 30° in the Opposite direction to compensate for the torsional strain produced (39). This asymmetric orientation puts the arginine finger in the optimal position for binding the y-phosphate in the neighboring A and B subunits, whereas in the C subunits it is too far away for binding (39). In the DnaB-like helicases, residues at the subunit interface of one monomer bind the nucleotide, but it is the arginine finger of the neighboring monomer that participates in ligating the y-phosphate (39,57). Our proteolysis data suggest that the human mtDNA helicase may also have an asymmetric orientation within the hexamer. In the T57/T50 heterohexamer, catalytically-competent hexamers would likely comprise oligomers in 42 which T57 occupies minimally the four neighboring subunits that are oriented to bind nucleotide. Although T50 is less abundant in our preparations, hexamers in which it neighbors T57 would be functionally inactive. We found that the N-terminal proteolytic product T28 lacks ATPase activity. T28 does not contain the classic Walker A and Walker B motifs located in conserved motifs I and H in SF -1 and SF-2 family members, as do N-terminal only domains of T7 gp4 and E. coli DnaB protein that exist as monomers under certain conditions (25,27,50,51,55). Clearly, the ATPase activity of the mitochondrial enzyme requires residues within the C- terminal domain. At the same time, similarities shared by T28 and the N-terminal fragments of T7 gp4 and DnaB protein may provide insight for its possible role in protein-protein interactions and/ or helicase regulation (56). Bacteriophage T7 gp4 and E. coli DnaB protein are members of the DnaB-like family of replicative DNA helicases (25,33,46). Here we probe the physical features of the mitochondrial enzyme and conclude that the hexarneric human mtDNA helicase is a modular protein comprising two N- and C-terminal structural domains. We also show that the essential residues for nucleotide hydrolysis, as well as those involved in oligomerization, reside exclusively in the C-terminal domain. Together with previous functional data, the human mtDNA helicase may be classified firmly within the DnaB- like family (43). 43 CHAPTER III COFACTOR EFFECTS ON THE SOLUBILITY, QUATERNARY STRUCTURE AND CONFORMATIONAL CHANGES OF THE HUMAN MITOCHONDRIAL DNA HELICASE 44 Summary We examined the effects of cofactors on the stability, oligomerization and conformational change of the human mtDNA helicase. The low salt-sensitivity of the mitochondrial enzyme can be stabilized by temperature, Mg2+ and nucleotide. We show through glutaraldehyde cross-linking that bound nucleoside triphosphate induces an increase in oligomerization beyond a hexarneric form. Using trypsin proteolysis, we demonstrate that nucleoside triphosphate binding results in a conformational change that differs from nucleoside diphosphate binding. Sequence homology of a phosphate sensor residue suggests this change may lead to a mechanism of DNA loading. We also find that DNA binds in a region homologous to the DNA binding loops of bacteriophage T7 gene 4 protein. Our studies reveal that the human mitochondrial DNA helicase shares basic properties with the SF-4 replicative helicases and may load DNA in a manner similar to other AAA+ ATPases. 45 Introduction In the absence of DNA, the stable, oligomeric state of helicases varies in accordance with the composition of their environment (69). Increasing temperature has been shown to provide stability to E. coli DnaB protein and T4 gp41 by promoting the multimerization of monomers (70,71). Similarly, the presence of nucleotide drives the T4 gp41 dimer to a higher oligomeric state (45). Hydrodynamic studies of E. coli DnaB protein reveal that the presence of Mg”, nucleotide or increasing salt induces the dimerization of trimers into a hexarneric form (44,70). T7 gp4 has been shown to exist as a dimer and trimer that oligomerizes into hexamers in the presence of dTTP, but in the absence of Mg2+ (33,34,72). Although hexamers are believed to be the active form of replicative DNA helicases during translocation, dodecamers have been identified that participate in multiple cellular processes, such as the initiation of replication, replication fork migration and branch migration (26,73-76). The thCM replicative helicase, a model archaeal enzyme for the study of eukaryotic chromosomal DNA replication, exists in this dodecarneric state and is believed to firnction at the origin of replication (66,77). A second well-studied dodecamer is the simian virus 40 large T antigen that melts and unwinds dsDNA at the origin during viral DNA replication (78). This protein uses an iris mechanism for melting DNA by pulling dsDNA into the double hexamer and extruding ssDNA through the side-wall channels of the dodecamer (79). This mechanism resembles the proposed twin pumping mechanism used by E. coli DnaB protein in which two helicases simultaneously melt dsDNA (75). All of these proteins can exist as hexamers, but for the described cellular mechanisms, a higher-order species is required. The binding of cofactors to helicases not only induces oligomerization of the 46 enzymes into a stable, active state, it also brings about a conformational change that is necessary for its function during replication (80). Circular dichroic spectra of E. coli DnaB protein shows a conformational change upon nucleotide binding (50). Similar findings reported by Romano et al. illustrate numerous conformations of T7 gp4 in the presence of different nucleotides and DNA (80). These conformations are presumed to facilitate nucleotide hydrolysis and the mechanism of ssDNA translocation that leads to dsDNA unwinding. In T7 gp4, Richardson et al. recently showed that the type of nucleotide present, di- or tri-phosphate, is detected by a phosphate sensor at histidine 465 that dictates a conformational switch from heptameric to hexarneric states, respectively. It is hypothesized that the loss of one subunit may be a mechanism of loading onto DNA (73). A fundamental property required of all replicative helicases is the ability to bind to ssDNA. A crystal structure of T7 gp4 confirmed electron microscopic studies by showing that C-terminal domain loops projecting into the center of the ring are responsible for ssDNA binding (35,39,49,81). This function is dependent on the hexarneric form of the T7 gp4 and the binding of nucleotides, but not Mg2+ (33,72,82). To date, although the substrate specificity in DNA unwinding has been examined, the DNA binding properties of the human mtDNA helicase have not been evaluated directly (43). We explored some basic characteristics of the novel mitochondrial enzyme by examining aspects common to E. coli DnaB-like helicases, and helicases that lie outside this group of enzymes. The stabilization of the human mtDNA helicase under physiological ionic conditions was examined, and we find that nucleotide is required for the retention of a soluble, stable protein in a low ionic environment. The oligomeric state, 47 in addition to conformational changes under the stabilizing conditions was also determined. This study advances the understanding of the oligomeric states and conformations of the human mtDNA helicase during its role in replication under physiological conditions, and reveals some unexpected physical aspects that suggest the possibility of other cellular functions. 48 Experimental Procedures Materials Trypsin, STI, adenosine 5’-['y-thio] triphosphate (ATPYS) was purchased from Sigma. Calf thymus DNA (highly polymerized Type I) was purchased from Sigma and was activated by partial digestion with DNase I (Boehringer Mannheim). SDS for general gel electrophoresis was from Pierce. Nitrocellulose membranes were purchased fi'om DOT Scientific, Inc. Rabbit antisera raised against recombinant human mtDNA helicase was prepared as described by Wang et al. (83). Horseradish peroxidase-conjugated anti- rabbit IgG was purchased from BioRad Laboratories. ECL Western blotting reagents were purchased from Amersham Biosciences. Methods Protein Overexpression and Purification of Human mtDNA Helicase-N- terrninally His-tagged human mtDNA helicase was produced by overexpression in Spodoptera frugiperda cells and purified to homogeneity, as described in Chapter 2. Solubility Assay- Reaction mixtures (25 p1) contained 35 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 50 pg/ml N-terminally His-tagged human mtDNA helicase in the absence/presence of four cofactors: 4 mM Mg“, 2 mM adenosine dinucleotide (ADP), 2 mM ATP, and 2 mM ATP'YS. Incubation was for 1 h at 4, 24 and 37° C followed by centrifugation at 10,000 rpm for 10 min. Soluble and insoluble fractions were made 1% in SDS, heated for 2 min at 100° C and electrophoresed in a 10% polyacrylamide gel. Protein bands were visualized by silver staining. The data were photographed and quantitated using Kodak 1D software. 49 Cross-Linking Analysis of the Human mtDNA Helicase- Reaction mixtures (25 pl) containing 5 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50 p g/ml of N-terminally His-tagged human mtDNA helicase were incubated for 30 min at 37° C in the absence or presence of four cofactors: 4 mM Mg”, 2 mM ADP, 2 mM ATP and 2 mM ATP'yS. Cross-linking with 0.015% glutaraldehyde was performed at 37° C after incubation and the reactions were quenched with 0.4 M Tris-HCl, pH 7.5. Samples were made 1% in SDS, heated for 5 min at 86° C and electrophoresed in a 4% polyacrylamide gel (16 X 15 X 0.5 cm) according to Laemmli (84). Proteins were transferred to nitrocellulose membranes and probed by immunoblotting. Protein 1mmunoblotting—Nitrocellulose filters were preincubated for 1 h with 5% skim milk in PBS, followed by incubation for 2 h with human mtDNA helicase antibody (1:500 dilution in PBS containing 0.1% Tween 20). Filters were washed four times with PBS containing 0.1% Tween 20, incubated for 1 h with horseradish peroxidase- conjugated anti-rabbit IgG (1 : 1500 dilution in PBS containing 0.1% Tween 20) and washed four times with PBS containing 0.1% Tween 20. Protein bands were visualized using ECL Western blotting reagents. T rypsin Digestion-Reaction mixtures (25 pl) contained 35 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 50 pg/ml N-terrninally His-tagged human mtDNA helicase were incubated for 30 min at 24° C in the absence or presence 1 mg/ ml calf thymus DNA and four cofactors: 4 mM Mg”, 2 mM ADP, 2 mM ATP and 2 mM ATP'yS. Trypsin was added to reactions at a helicase:trypsin ratio of 1:1, followed by incubation for 10 min at 24° C. Digestions were stopped by the addition of STI at a trypsin:STI ratio 50 of 1:2. Digestions products were separated using SDS-PAGE, and analyzed by silver staining. Other Methods-Gel electrophoresis and protein transfer were performed as described by Wang et al. (83). 51 Results Factors stabilizing the human mtDNA helicase under physiological ionic conditions. The purified hexarneric human mtDNA helicase is a soluble, stable polypeptide at 330 mM NaCl (Chapter 2, Fig 1, A and B). To identify factors that might stabilize the human mtDNA helicase at moderate (150 mM NaCl) and low (100 mM NaCl) salt concentrations, we incubated the protein at different temperatures in the presence of various cofactors such as Mg2+ and nucleotide, and examined the stability of the enzyme by evaluating its retention in the soluble fraction following centrifugation (see Methods). In the absence of cofactors and moderate salt, temperature increasing from 4-37° C help retain the protein in a soluble state, whereas at low salt, complete insolubility is observed (Fig. 7, A-C). At both salt concentrations and with temperatures increasing from 4-37° C, the presence of Mg2+ increases the solubility of the protein. Stability is further increased with the addition of ATP. In contrast, the solubility is slightly decreased by the addition of ADP at conditions of low ionic strength. The greatest effect on solubility is in the presence of Mg2+ and the non-hydrolyzable ATP analogue, ATPyS. We conclude that in a physiological ionic environment, the human mtDNA helicase is most stable when nucleoside triphosphate (NT P) is bound. 52 Fig. 7 Factors stabilizing the human mtDNA helicase under conditions of physiological ionic strength. Human mtDNA helicase was incubated at 150 mM and 100 mM NaCl in the absence (white bars) and presence of Mg” (black bars), Mg2+-ATP (dark gray bars), Mg2+-ADP (medium gray bars), Mg2+-ATPyS (light gray bars) and subjected to centrifugation. Soluble samples at 4° C (A), 24° C (B) and 37° C (C) were analyzed by SDS-PAGE followed by silver staining. 53 > Solubility, % w Solubility, % O Solubility, % 150 mM NaCl 100 mM NaCl Figure 7. Factors stabilizing the human mtDNA helicase under physiological ionic conditions 54 Oligomeric states of the human mtDNA helicase in the presence of cofactors. Glutaraldehyde cross-linking was used to examine the oligomeric state of the human mtDNA helicase in the presence or absence of cofactors (Fig. 8). In the absence of glutaraldehyde (lanes 1 and 2), a dimeric form of the protein is stable even under denaturing conditions. In the presence of glutaraldehyde and with or without cofactors, the protein exists in multiple oligomeric states ranging from monomer to heptamer (lanes 3-11). It is evident that the abundance of each band increases with the addition of cofactors (lanes 5-11). The most prominent oligomeric species are observed in the presence of Mg2+ and ATP'yS (lanes 9 and 10). The addition of ATP'yS with or without Mg2+ (lanes 7-10) produces an oligomer beyond heptamer, and may be that of a double hexamer. 55 Fig. 8 Oligomeric states of the human mtDNA helicase in the presence of cofactors. Hmnan mtDNA helicase was pre-incubated under the indicated conditions for 30 min at 37° C prior to incubation with 0.015% glutaraldehyde for 20 and 40 min at 37° C. Samples were analyzed by SDS-PAGE followed by enhanced chemiluminescence. Lanes 1-2, absence of glutaraldehyde; lanes 3-11, presence of glutaraldehyde under the following conditions: lanes 3-4, absence of cofactors; lanes 5-6, Mg2+ only; lanes 7-8, ATPyS only; lanes 9-10, Mg2+-ATPyS; lane 11, Mg2+-ADP. Molecular weight standards were run in an adjacent lane. 56 _ ‘ high order ' - oligomer - heptamer - hexamer — pentamer _ I }dimer - monomer Figure 8. Oligomeric states of the human mtDNA helicase in the presence of cofactors 57 Trypsin digestion of the human mtDNA helicase reveals conformational changes upon cofactor and substrate binding. To demonstrate the conformational changes induced by the binding of cofactors, such as Mgr", nucleotide or DNA, we proteolyzed the human mtDNA helicase under different conditions (see Methods) and compared the patterns of digestion produced (Fig. 9). Magnesium alone or Mg2+-ADP (lanes 3 and 9), respectively produce the same pattern as in the absence of cofactors (lane 2) and therefore do not appear to induce a conformational change. However, the addition of ATP or ATP’yS prevents the production of the lower molecular weight proteolytic products T30 and T28 that are produced in the absence or presence of Mg2+ and Mg2+-ADP (lanes 4 and 5). In the presence of Mg2+ and DNA and (lane 6), the intermediate proteolytic products T50 and T44 are absent, whereas the addition of NTP (lanes 7 and 8) results in a slight retention of T44. These results suggest that NTP and DNA either bind regions near the trypsin cleavage sites, or produce a conformational change that renders the trypsin sensitive sites inaccessible. 58 Fig. 9 Trypsin digestion of the human mtDN A helicase reveals conformational changes upon cofactor and substrate binding. Human mtDNA helicase was proteolyzed with trypsin at 100 mM NaCl at 24° C. Samples were analyzed by SDS-PAGE followed by silver staining. Lane I , undigested helicase; lane 2, absence of cofactors; lane 3, Mg“ only; lane 4, Mg2+ -ATP; lane 5, Mg2+-ATPyS; lane 6, Mg 2* - DNA; lane 7, Mg 2+-ATP-DNA; lane 8, Mg 2* -ATPyS-DNA; lane 9, Mg 2* -ADP. 59 mtDNA helicase — fl trypsin -— _,72 - 66 - 57 _ so —44 _/34 -—-'_3o -"-28 Figure 9. Trypsin digestion of the human mtDNA helicase reveals conformational changes upon cofactor and substrate binding 60 Discussion The fluctuating ionic environment within the matrix of the mitochondrion varies in accordance with the metabolic state of the organelle, and is likely involved in mitochondrial regulation (85). Our hexarneric recombinant human mtDNA helicase, however, is purified as a stable, soluble recombinant protein at a salt concentration of 330 mM NaCl (Chapter 2, Methods and Fig 1. A and B). At ionic conditions more representative of the mitochondrial environment, we found that in the absence of cofactors, such as Mg2+ and nucleotide, the enzyme is largely insoluble. Higher salt concentrations along with increasing temperature cause an increase in solubility and therefore, produce a stabilizing effect on the human mtDNA helicase. This is consistent with studies of E. coli DnaB protein, T7 gp4 and T4 gp41 that show protomer multimerization as temperature or ionic strengths are elevated (44,71,86). Mg2+ has a moderately stabilizing effect, and is slightly destabilized by the addition of nucleoside diphosphate. The mtDNA helicase is most stable when Mg2+ is present and NTP is bound. Our cross-linking data show that in the absence of glutaraldehyde and cofactors, a dimeric form exists that possesses very strong subunit interactions and is resistant to the conditions of denaturing gel electrophoresis. Therefore, the previously demonstrated hexarneric state of the human mtDNA helicase could assemble from a trimerization of dimers, as shown for recombinant forms of T4 gp41 and simian virus 40 large T antigen (45,79). We found that, in the absence of cofactors, the abundance of the multiple oligomeric bands is very low, corroborating with our solubility data. The addition of Mg2+ produces an increase in the band intensity that we interpret as a stabilizing effect on the hexamer, and is consistent with cross-linking studies of E. coli DnaB protein (33). 61 Band density firrther intensifies in the presence of NTP and coincides with the increase in solubility under the same condition. We believe that the presence of cofactors prevents the dissociation of hexamers into unstable, lower order species. Furthermore, the addition of Mg2+ and NTP induces oligomerization to a state beyond hexamer/heptamer and may represent a double hexamer. Our data show that the N-terminus of the human mtDNA helicase has a positive effect on nucleotide hydrolysis by the full-length enzyme (Chapter 2, Fig. 6). Therefore, it is possible that this effect is due to the participation of this region in the multimerization of the enzyme into a double hexamer. A crystal structure of the N- terminal domain of thCM was found to be responsible for its oligomerization into a double hexamer (Fig. 10) (74). Furthermore, the crystal structure of simian virus 40 large T antigen, a modular helicase capable of double hexamer formation, reveals a hexarneric protein that readily dissociates into monomers in the absence of NTP but is subsequently stabilized and stimulated to hexarnerize with the addition of ATP (79). Kaplan et al. have shown that two hexamers of E. coli DnaB protein on opposite sides of a four-way DNA junction function in branch migration (75). This, in combination with the dimerization ability of its N-tenninal domain, suggests that the hexamers may form a dodecarneric complex (56). The existence of the hmnan mtDNA helicase in this multimeric state opens up a broad range of potential roles for the mitochondrial enzyme which are known for the other AAA+ ATPases, a family of proteins that participate in a broad range of cellular functions including recombination and DNA repair. 62 Fig. 10 Structure of the thCM double hexamer. Final 3D reconstruction of the thCM structure in a view illustrating different orientations (A), a View demonstrating N-terminal interactions resulting in dodecamerization (B), and a view showing density of the central section (C). 63 my: lmLM.wl Gomez-Morena, Y., dd. J Biol Chem (2005) 280: 40909-40915 Figure 10. Structure of the thCM double hexamer We show by trypsin digestion that the human mtDNA helicase in the NTP bound state undergoes a conformational change; this is commonly seen in the replicative helicases E. coli DnaB protein, T7 gp4 and T4 gp4], facilitating their interactions with ssDNA (50,70,71,80). In T7 gp4, this conformational change was found to be a switch from a heptamer to a hexamer in the presence of NTP (73). In the absence of nucleotide, T7 gp4 was crystallized as a heptamer, a quaternary structure that is accommodated by different inter-subunit contacts in a region between the linker and helicase domain (58). This region in T7 gp4 corresponds to the cleavage site of the htunan mtDNA helicase, R371, which results in the production of T30. Our proteolytic data shows that T30 is produced only in the absence of cofactor, the presence of Mg2+ and when nucleoside diphosphate is bound. We assume that, under these conditions, the human mtDNA helicase is predominantly a heptamer. Therefore, when NTP is bound and the T30 cleavage site is no longer accessible, the enzyme exists predominantly as a hexamer, and potentially as a double hexamer. These oligomeric states are consistent with the conditions under which the T7 gp4 loses a subunit and switches from heptamer to hexamer to form an open complex (73). The loss of one subunit has been demonstrated in electron microscopic studies of other helicases such as T4 gp41, Thermus thermophilus HB8 RuvB and thCM and is believed to be a mechanism for DNA loading (73,74,76,87,88). In addition, the phosphate sensor at H465 revealed by Crampton et a1. is conserved in the human mtDNA helicase as well and may serve a similar firnction (73). We did not demonstrate directly that the presence of NTP induced a switch from heptamer to hexamer in the mtDNA helicase, but our proteolysis data provide indirect evidence, and suggest that the enzyme may undergo a similar conformational change, and also share a similar mechanism for loading around DNA. 65 The cleavage site that results in the production of T28, aa 405, is consistent with nucleotide binding within conserved helicase motif 1, also known as the Walker A motif, which participates in the binding of nucleotide in all helicases (Chapter 2, Fig. 3A). Our data show that, when NTP is bound, this region is protected from trypsin attack and results in a loss of T28. We found by limited proteolysis that DNA binding occurs in the presence of Mg2+ alone. This finding is similar to that observed with thCM, but contrasts with the nucleotide-dependent DNA binding of E. coli DnaB protein and T7 gp4 (66,70,82). Taken together with the existence of a potential double hexamer, we suggest a possible role for the human mtDNA helicase in functions that require DNA binding but not translocation, such as dsDNA melting during the initiation of replication as shown for thCM and simian virus 40 large T antigen (66,89). T7 gp4 binds DNA through three DNA binding loops located within the C-terrninal domain (Fig. 11A) (39). We found that the proteolytic products T50 and sometimes T44 are no longer produced when DNA is present. As shown previously, T50 is produced from a cleavage occurring C-terrninal to motif IV and based on gel mobility, T44 would predictably result from a cleavage between motif III and motif IV (Fig. 113). The crystal structure of T7 gp4 shows that these loops into the center of the ring and contact ssDNA (Fig. 11C) (39). The data reported here suggest that the mitochondrial enzyme binds DNA in a homologous region of T7 gp4, as evident by the protection of these areas in the presence of DNA. 66 Fig. 11 DNA binding loop homology between T7 gp4 and the human mtDNA helicase. (A), Schematic illustrating the location of DNA binding loops of T7 gp4. (B), Schematic illustrating T50 and T44 cleavage sites. (C), Model illustrating T7 gp4 DNA binding loops projecting into the center of the ring. 67 A Bacteriophage T7gp4 Primase Helicase Domain Linker Domain Motif 1 2 3 4 5 6 1 1a 2 3 4 L+ DNA Binding Loop. B N-terrninal region linker C-tcrminal region 4 ¥ 4 ‘ r ‘ r 1 I IA II III IV 684aa mnDNAhencase I m T66 :43 4340: T57 145 764°: 4 ~591 T50 ‘1 5 l m l ——————————————————————————— I 45 ~45 T34 l Q. ~640 T30 I72 J. 4 ~406 T28 ll 5 l C ssDNA translated from submit to l “I? II 333cm: swunll E Loop ll 2 ‘ x t ‘7— ’ T T _._ . I Helicase ‘ '2 5 subunit Y , mp t . "’ 1, . .' ‘. l"- ‘ i l :—- ' -;‘-' , dTDP+Pl ‘ fr" t , 0 '§ If, i‘ ‘ -‘~., ,- l. ' . . ‘ i I ‘r 5 Crampton, D., et al. Mol. Cell (2006) 21:165-174 Figure 11. DNA binding loop homology between T7 gp4 and human mtDNA helicase 68 In summary, we have examined the effects of cofactors on the solubility, multimerization and conformation of the human mtDNA helicase. Although our findings are generally consistent with those of other E. coli DnaB-like family members, certain differences, such as oligomeric changes in the presence of NTP and requirements for DNA binding, suggest potential roles for the mitochondrial enzyme that may lie outside the reahn of replication. The human mtDNA helicase has a bacterial ancestry that is apparent by its sequence similarity, physical properties and biochemical characteristics shared with T7 gp4. However, our data argues that the human mtDNA helicase also resembles other eukaryotic helicases, and suggests functional diversity from its associated family members. Similarly, archaeal organisms share genetic characteristics with both prokaryotic and eukaryotic systems, and although their proteins are more related in sequence to bacteria, their cellular firnctions are more similar to eukaryotic processes (90,91). It is reasonable to assume that through evolution an ancestral, bacterial protein such as the human mtDNA helicase would adopt mechanistic features of its eukaryotic host to become a more efficient cellular machine. Therefore, we must look beyond the bacteriophage and bacterial systems for information regarding the importance of this novel helicase in mitochondrial biogenesis. 69 CHAPTER IV SUMMARY AND FUTURE PERSPECTIVES 70 Summary This study has revealed physical and functional aspects of the human mtDNA helicase. Physically, we can confirm its hexarneric, modular architecture and propose an asymmetric orientation. We have determined that the region responsible for maintaining a stable oligomer lies between a 405-590. Magnesium and temperature have a stabilizing role in oligomerization by preventing the dissociation of hexamers/heptamers into monomers. The binding of nucleoside triphosphate induces a conformational change in the quaternary state beyond the hexamer/heptamer oligomer and may be that of a dodecamer. Physical oligomeric states may switch depending on the type of nucleotide bound and may lead to a mechanism for DNA loading. Either ssDNA or dsDNA binding occurs in a region homologous to the DNA binding loops of T7 gp4 that are located within the center of the ring structure. Functionally, we find that the N- and C-termini of the mtDNA helicase have differential effects on nucleotide hydrolysis, and that this activity resides in the C- terminal domain of the enzyme. Nucleotide binds in motif 1, consistent with the role of the Walker A motif in all enzymes possessing helicase activity. We have identified R609 to be a putative arginine finger involved in the hydrolysis reaction. We hypothesize that the positive regulatory effect of the N-terrninus may be due to a role in higher-order oligomer formation. The human mtDNA helicase is a novel enzyme with numerous physical and functional aspects still awaiting discovery. Its link to a human mitochondrial disease causing external eye muscle paralysis warrants study of this enzyme. The features identified here could help expand the present perceptions of protein functions within the mitochondria and mitochondrial biogenesis. 71 Future Perspectives This study contributes to the growing biochemical data available for the human mtDNA helicase, and lays the groundwork for further investigation. The modular architecture of the enzyme suggests segregation of regions responsible for either physical or functional properties, or both. Specific properties have been assigned to distinct regions of other helicase enzymes such as T7 gp4 or E. coli DnaB protein and need to be assessed in the human mtDNA helicase. These include protein-protein interaction sites, and residues involved in oligomerization, nucleotide and DNA binding. Studying these functional attributes within the N- and C-terrninal domains will clarify further the roles of both regions. Furthermore, the high degree of similarity with T7 gp4 in both secondary structure and response to protease treatment suggest that a primase activity may reside in the N-terminal domain. To demonstrate potential primase activity, the production of a recombinant form of the human mtDNA helicase containing a mutation at E445 which is homologous to the catalytic residue E343 in T7 gp4 could be used. In T7 gp4, this residue is known to be important for hydrolysis and results in an enzyme that has lost its ability to translocate and therefore binds tightly to ssDNA (40). A primase assays on the recombinant enzyme that is capable of strong DNA interaction could help confirm or refute this hypothesis, and contribute to our understanding of the mitochondrial replisome. Investigating the cellular firnction of the putative higher order oligomer identified here may reveal activities outside of DNA replication per se. The potential involvement of the N-termini in this state may determine a regulatory role for the termini during fork advancement. Our present data demonstrates a functional role for both termini in nucleotide hydrolysis, but how this relates to helicase function is unclear. One possible 72 regulatory role for the the termini could be during intitation of replication, a highly regulated step. During this step, NTP binding simultaneously leads to dsDNA origin binding and enzyme dodecamerization through the N-terminus. The C-terrninus negatively effects hydrolysis and prevents translocation of the dodecamer, and thereby allowing dsDNA melting at the origin. Subsequent cleavage of both termini by a protease could result in the loss of dodecamerization, an increase in hydrolysis and translocation of two hexamers in opposite directions. To test this hypothesis cross- linking of the human mtDNA helicase lacking its N-terrninus will investigate whether this region is required for dodecamer formation as shown for thCM (77). Also, helicase assays of truncated forms lacking either the N- or C- terminus only will demonstrate translocation abilities compared to the full-length enzyme. In a hypothesis such as this, the production of a proposed protease would be the regulator of initiation and could fluctuate in accordance with the metabolic state of the mitochondria. This theory would be supportive of the strand-coupled model of human mtDNA replication. Furthermore, evaluation of protein-DNA interactions and nucleotide stabilization remain potential avenues for exploration to clarify the positive and negative effects on the hydrolysis activity. DNA binding by the human mtDNA helicase was addressed indirectly here, but needs further clarification such as whether ss- or dsDNA was bound. Proteolytic digestions in the presence of both types of substrate followed by comparative analysis of digestion patterns would discriminate substrate specificity. The differential pattern observed with and without nucleotide in the presence of DNA indicates different conformations that might correspond to various oligomeric states. Cross-linking studies in the presence of DNA will address this issue. A nucleotide-induced quaternary switch 73 could also be examined by crystallography and in electron microscopy, and could elucidate the proposed DNA loading mechanism. A crystal structure may also confirm or disprove the proposed asymmetry in the hexamer, and illustrate potentially conserved features consistent with other replicative helicases such as ring structure, nucleotide binding at the subunit interface, as well as demonstrate the DNA binding loops projecting in the center of the ring. To evaluate thoroughly the DNA binding activity of the mitochondrial enzyme, the size of DNA bound under various oligomeric states should be evaluated and could shed light on the roles of the human mtDNA helicase within the cell. 74 List of Publications——Tawn D. Ziebarth Ziebarth, T.D., Farr, C.L., and Kaguni, LS. (2006) Modular architecture of the hexarneric human mitochondrial DNA helicase. In preparation for J. Mol Biol. Ziebarth, T.D., and Kaguni, LS. (2007) Cofactor effects on the solubility, quaternary structure and conformational changes of the human mitochondrial DNA helicase. In preparation for J. Biol. 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