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Nucleoside Diphosphokinase of Eschericia Coli and Its Interactions With

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Nucleoside Diphosphokinase of Eschericia Coli and Its Interactions With AN ABSTRACT OF THE THESIS OF Nancy Bisset Ray for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on May 8, 1992 Title : Nucleoside Diphosphokinase of Eschericia coli and its Interactions with Bacteriophape T4 proteins of DNA synthesis Redacted for privacy Abstract approved: Escherichia coli nucleoside diphosphokinase (NDPK), the product of gene ndk, synthesizes nucleoside triphosphates from the corresponding diphosphates. This bacterial enzyme is an integral component of the T4 bacteriophage dNTP synthetase complex, a multienzyme complex for deoxyribonucleotide biosynthesis, and it plays an indispensable role in T4 DNA replication. A goal was established to locate and clone ndk, in order to overexpress the gene and obtain large enough quantities of the enzyme for the analysis of protein interactions, involving NDPK and proteins of both the T4 dNTP synthetase complex and the T4 DNA replication complex. NDPK was first purified 5000-fold from crude extracts of E. coli B cells for N-terminal amino acid sequencing. Over forty residues of N-terminal sequence were determined, providing information used to design mixed oligonucleotide probes, designed to search for the ndk gene in the Clarke and Carbon E. coif ColE1 plasmid library. A 3.2-kb Pst I fragment from the Clarke and Carbon plasmid, pLC34-9, hybridized specifically to one of the probes and was subcloned into pUC19. Six- fold higher NDPK enzyme activity, over host NDPK enzyme activity, was generated by the recombinant pUC19 plasmid in JM83 cells. A 6.0-kb EcoRl fragment from the Kohara E. co/i lambda library, mapping to approximately the same area, was also cloned into pUC18 based on the elevated, overlapping NDPK enzyme activity of two lambda clones, 2D5 and 7F8. NDPK was overexpressed from a pUC9 recombinant plasmid obtained from Masayori Inouye, and purified to homogeneity for protein-protein interaction studies with T4 early proteins (proteins primarily involved in T4 dNTP metabolism and DNA replication). NDPK was then immobilized onto an affinity column and extracts of [35S1-methionine-labeled, T4-infected and uninfected E. coil B cells were applied to the column. Labeled proteins were then eluted from the column with increasing concentrations of salt and analyzed by both one and two-dimensional polyacrylamide gel electrophoresis. Several T4 dNTP biosynthesis enzymes, including dihydrofolate reductase, the large and small subunits of ribonucleotide reductase, and dCMP hydroxymethylase, as well as several T4 DNA replication proteins, were bound to the column at 0.2 M NaCI, and eluted at 0.6 M NaCI. Protein-protein interactions involving NDPK and T4 early proteins have also been investigated by NDPK-anti-idiotypic antibody immunoprecipitation analysis. One T4 early protein has been found to be specifically immunoprecipitated by NDPK-anti-idiotypic antibody. This protein is not among those so far identified by two-dimensional polyacrylamide gel analysis, and further identification is underway. The specific interaction of NDPK and T4 DNA polymerase has also been investigated through the use of fluorescence anisotropy. Nucleoside Diphosphokinase ofEscherichia coliand its Interactions with Bacteriophage T4 Proteins of DNA Synthesis by Nancy Bisset Ray A Thesis submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed May 8, 1992 Commencement June 1993 APPROVED: Redacted for privacy Professor of Biochemistry and Biophysics in chargeof major Redacted for privacy Head of department of Biochemistry and Biophysics Redacted for privacy Dean of GraduateiVhool Date thesis is presented May 8, 1992 Typed by Nancy B. Ray for Nancy B. Ray Acknowledgements I would like to thank my advisor, Christopher K. Mathews, for the advice and support he has provided throughout my graduate studies.I would also like to thank Gerry Lasser and Linda Wheeler for allowing me to benefit fromtheir expertise. I greatly appreciate the advice and assistance I received from Dr.Sonia Anderson and Dr. Dean Malencik, and would like to extend special thanks to Anne Carr and Tanya McCoy for their technical assistance. I would like to convey my sincerest appreciation to my husband,William Anthony Cummins, who has been very patient and supportive throughout my graduate career, and who provided invaluable computer expertise. TABLE OF CONTENTS I. Introduction 1 A. General Introduction 1 B. General Description of Bacteriophage T4 4 C. The T4 dNTP Synthetase Complex and its Relationship to T4 DNA Replication 5 D. NDPK: Historical Background 11 E. NDPKs Reaction Mechanism and Physical Properties 14 F. The Role of NDPK in the T4 dNTP Synthetase Complex and in T4 DNA Replication 15 G. Other Detected Protein Interactions Involving NDPK 19 H. Strategy for Cloning and Overexpressing the ndk Gene 20 I.Significance of Cloning and Overexpressing the ndk Gene 24 J. Other E. coli GTP-binding Proteins 25 K. Homology Among NDPK Sequences 26 L. NDPK: Its Relationship to the Tumor Suppressor Nm23 Gene and to Drosophila Development 29 M. Current Work 32 II.Materials and Methods 35 A. Materials 35 B. Media, Antibiotics and Buffers 35 1. Media 35 2. Antibiotics 37 3. Buffers 37 C. Cells, Plasmids and Phage 39 D. Purification of NDPK from Crude Extracts of E. coli B Cells 39 1. Preparation of Extract and Salt Precipitations 39 2. DEAE Sephadex and Hydroxyapatite Fractionations 41 3. Blue Sepharose (Matrex Blue) Fractionation 41 4. Chromatofocusing Fractionation 41 5. Molecular Sieve Fractionation 42 6. HPLC Fractionation 42 E. Enzyme Assays 42 F. Protein Determinations 43 G. Gel Electrophoresis 45 1. One-dimensional SDS PAGE 45 2. Two-dimensional PAGE 45 3. DNA Agarose Electrophoresis 47 4. DNA Acrylamide Electrophoresis 47 H. N-terminal Amino Acid Sequencing 48 I.Growth and Amplification of Clarke and Carbon ColE1 and Kohara Lambda Clones 48 1. Clarke and Carbon Clones 48 2. Kohara Lambda Clones 49 J. Plasmid, Phage, and Single-stranded DNA Preparation and Analysis 49 1. Plasmid DNA 49 2. Phage DNA 50 3. Single-stranded DNA 51 K. Mixed Oligonucleotide Probes 52 1. Design and Synthesis 52 2. Determination of Purity 52 L. Southern Blotting 53 1. Southern Transfer 53 2. End-labeling of Oligonucleotide Probe 53 3. Prehybridization 54 4. Hybridization 54 5. Wash 55 6. Development 55 M. Cloning 55 1. DNA Isolation from Agarose Gels 55 2. Ligation 56 3. DNA Transformation 57 N. DNA Sequencing 58 1. Plasmid Sequencing (double-strand) 58 2. M13 Sequencing (single-strand) 59 0. Overexpression of Recombinant NDPK 59 P. Purification of Overexpressed NDPK 60 1. Preparation of Extract and Salt Precipitations 60 2. Blue Sepharose Fractionation 61 3. MonoQ Fractionation 61 Q. Characterization of NDPK 61 1. UV Scan 61 2. Quantitation of Amino Acids 62 3. Intrinsic Fluorescence Scan 62 R. Immobilized NDPK Affinity Chromatography 63 1. Immobilization of NDPK 63 2. Preparation of Extract 63 3. Affinity Column Fractionation 64 4. Two Dimensional Gel Analysis 66 S. Antibody Generation and Fractionation 67 1. Antibody to Denatured NDPK 67 2. Antibody to Native, Active NDPK 67 3. Protein A Fractionation of Antibody to Denatured NDPK 68 4. Fractionation of Antibody to Native, Active NDPK 68 T. Western Blotting 69 U. Immunoprecipitations 70 1. Preparation of Extract and Precipitation 70 2. Identification of Immunoprecipitating Protein 71 V. Fluorescence Anisotropy Interaction Studies 71 1. Labeling of NDPK with 1,5-IAEDANS 71 2. Anisotropy Measurements 72 III.Results 73 A. Purification of NDPK from Crude Extracts 73 1.First Purification 73 2. Second Purification 75 B. NDPK N-terminal Amino Acid Sequence 78 1. Use in Design of Mixed Oligonucleotide Probes 78 2. Comparison to the M. xanthus N-terminal Amino Acid Sequence 83 C. Southern Blotting and Activity Assays with Clarke and Carbon Plasmids 85 D. Subcloning of the 3.2-kb Pst I Fragment and Six-fold Higher Activity 89 E. DNA Sequencing 89 F. NDPK Activity Assays of the Kohara Lambda Clones 91 G. Subcloning of the 6.0-kb EcoR1 Fragment 92 H. Improved Overexpression of NDPK from pKT8P3 92 I.Purification of Overexpressed NDPK 93 1. Salt Precipitations 93 2. Blue Sepharose Fractionation 95 3. MonoQ Fractionation 95 J. Characterization Studies 98 1. UV Scan 98 2. Quantitation of Amino Acids 98 3. Fluorescence 100 K. Immobilized NDPK Affinity Chromatography 102 1. Elution of Radioactivity 102 2. One-dimensional Analysis of Eluted Proteins 102 3. Two-dimensional Analysis of Eluted Proteins 106 L. Anti-idiotypic Antibody Interaction Studies 110 1. Immunoprecipitation with Anti-idiotypic Antibody to Denatured NDPK and Analysis 110 2. Two-dimensional Analysis of Immunoprecipitated Protein 115 3. Immunoprecipitation with Anti-idiotypic Antibody to Native, Active NDPK 119 M. Fluorescence Anisotropy Studies 119 1. Labeling with 1,5-IAEDANS 119 2. Anisotropy Measurements of NDPK and T4 DNA Polymerase 122 IV. Discussion 123 A. NDPK and Other E.coli GTP-binding Proteins 123 B. Potential Regulation of NDPK 125 C. NDPK's Role in T4 DNA Replication 127 D. Potential Interaction of NDPK and T4 DNA Polymerase 130 E. Unidentified Immunoprecipitated Protein 131 V. Bibliography 133 LIST OF FIGURES I-1. T4 dNTP Metabolism Pathway 3 1-2. Putative Coupling of the T4 dNTP Synthetase Complex and the T4 DNA Replication Complex 9 1-3. Kinetics of Thymidine Nucleotide Accumulation After Inhibition of T4 DNA Replication 16 1-4. Sensitivity of T4 Synthesis to Inhibition by Desdanine, an inhibitor of NDPK 18 1-5. Escherichia coil Chromosomal Map of Clarke and Carbon Plasmids 21 1-6. Escherichia col/ Chromosomal Map of Kohara Clones 23 1-7. Three Consensus Amino Acid Sequences of GTP-binding Proteins 27 1-8. NDPK Amino Acid Sequence Homology 30 1-9. Putative Role of NDPK/NM23 in the Activation of G Proteins 33 11-1. Coupled Enzyme Reactions of the Spectrophotometric Assay of NDPK 44 111-1. Purification Scheme Used in the Purification of NDPK from Crude Extracts of E.
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