AN ABSTRACT OF THE THESIS OF Mark Aguirre Bernard for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on December 16, 1998. Title: Host Kinases Involved in DNA Precursor Biosynthesis during Bacteriophage T4 Infection. Abstract approved: Redacted for privacy Christopher K. Mathews Although the Escherichia coli host has almost all of the enzymes necessary to synthesize nucleotides needed for bacteriophage T4 DNA replication, phage genes expressed early in infection encode enzymes for de novo DNA precursor biosynthesis and salvage from degraded host DNA. Eight early enzymes and two host enzymes comprise the multienzyme dNTP synthetase complex. The complex utilizes two host kinases for phage replication:nucleoside diphosphate kinase (Ndk) and adenylate kinase (Adk). The dNTP synthetase complex and the replication apparatus interact in vivo. dNTP synthesis is kinetically coupled to T4 DNA synthesis in a wild-type host but not in an ndk host. Moreover, one indirect and four direct experimental approaches demonstrate partial reconstitution of interactions between the two complexes in vitro. These interactions include Ndk and T4 DNA polymerase, which catalyze consecutive metabolic steps (dNTP synthesis and replication). The effect of the ndk mutation on the E. coli host was also studied. Disruption of the host ndk gene has been reported to cause a mutator phenotype due to nucleotide pool imbalances from a hugely increased dCTP pool.The pool imbalance has little effect on growth rate, since the ndk strain growth rate is 5.8 min. (15%) slower. However, the rate of phage DNA synthesis is reduced by 83.7% in the ndk strain relative to its parent. Although Adk complements ndk disruption, Adk's NDP kinase activity has high KM valuesfor dNDPsubstrates. Adk hasanovel (dNTP:AMP) phosphotransferase activity which synthesizes the dNTP's for replication. As measured by selectivity (kca/KM), Adk's (dNTP:AMP) phosphotransferase activity not its NDP kinase activityis the physiologically relevant mechanismfor dNTP synthesis in the ndk mutant.Therefore, ADP is the physiological phosphate donor instead of ATP. The ndk strain was found to have abnormally elevated nucleotide pools of UTP, CTP, dCDP and dTTP. In the ndk mutant, a high activity of Adk upon UDP leads to excess UTP production.Accompanied by a 3.2-fold increase in CTP synthetase activity, subsequent nucleotide pools are also increased, culminating in 18-fold dCTP and 2.4-fold dTTP pool accumulations, and hence, the mutator phenotype. C Copyright by Mark Aguirre Bernard December 16, 1998 All Rights Reserved Host Kinases Involved in DNA Precursor Biosynthesis during Bacteriophage T4 Infection by Mark Aguirre Bernard A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented December 16, 1998 Commencement June 1999 Doctor of Philosophy thesis of Mark Aguirre Bernard presented on December 16, 1998 APPROVED: Redacted for privacy Major Professor, representing Biochemistry and Biophysics Redacted for privacy Head of Department of Biochemistry and Biophysics Redacted for privacy Dean of Graduk School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for privacy Mark Aare Bernard, Author ACKNOWLEDGMENTS First, I would like to thank my family for their love and support. I especially would like to thank my wife, Tanya for her steadfast love, patience, courage under fire, encouragement and assistance. I am grateful to my parents, Joel and Beverly Bernard, whose consistently exemplary and honorable character has been inspirational and indispensable. I would also like to thank my brothers Stephen, David and Francis for all the good times and all their help.I thank my best friend, Eric Peterson, for his encouragement and for sharing his knowledge of physics and electronics. I would also like to thank members of the Mathews lab.I thank Dr. Christopher K. Mathews for the privilege of his impressive teaching and scholarship and for supporting this research.Thanks go to Dr. Stephen Hendricks particularly concerning nucleotide pool assays and to Dr. Indira Rajagopal, Dr. Ronald McClard, and Dr. Wayne Thresher for useful discussions.I thank Dr. Nancy Ray for her work on nucleoside diphosphate kinase and for the (in)famous anti-idiotypic antibody to Ndk. I also thank Cho Byung-Chul for purified gene 32 protein. I would like to thank Dr. Patrick Young and Dr. Ralph Davis especially for teaching their antibody techniques. I would also like to thank people from Beckman Instruments, Inc. for technology that proved useful in my Graduate School career. I thank Dr. Yuh-Nan Lin and Lynette Schneider for introducing me to immunochemistry.I would also like to thank Dr. Jack Zakowski, Dr. Ken Pierre and Mike Metzler for showing the coupled enzymatic assay method.I am also grateful to my former Department Director, Dr. Patricia Antonoplos, for her encouragement and her example of effective leadership. This research was supported by NSF grant MCB 96 06384 to Dr. Christopher K. Mathews. CONTRIBUTION OF AUTHORS Dr. Christopher K. Mathews provided intellectual support and mentorship, which were vital contributions to this research. Three people ransophisticated and lengthyanalytical procedures that contributed to this investigation. Stephen Hendricks performed nucleotide pool analysis from cell extracts by boronate column and anion exchange HPLC. Linda Wheeler kindly ran two-dimensional gels of T4 proteins. Barbara Revere (Center for Gene Research and Biotechnology, Oregon State University) sequenced purified Escherichia coli adenylate kinase and the 43 -kDa T4 protein immunoprecipitated by anti-idiotypic antibody to E. coli nucleoside diphosphate kinase. Recombinant DNA constructs and E. coli strains were also important to completing this research. Veronique Perrier and Dr. Octavian Barzu (both from the Pasteur Institute) provided plasmids for overexpressing E. coli adenylate kinase. Dr. Mark Young (University of Oregon) and Dr. William Konigsberg (Yale University) provided the plasmid for overexpressing T4 DNA polymerase. Dr. Qing Lu and Dr. Masayori Inouye(both from RutgersUniversity)provided theplasmidfor overexpressing E. coli nucleoside diphosphate kinase, as well as the series of E. coil strains with insertionally inactivated pykA, pykF or ndk genes. Collaborators also donated purified proteins and antibodies. Stephen Hendricks provided purified T4 ribonucleotide reductase (NrdA and NrdB). Steve Hendricks and Cho Byung-Chul provided one preparation each of purified T4 single-stranded DNA binding protein (gene 32 protein). Hui-Yun Zhang and Jake Zelenka each provided some of the NDP kinase used in this work. Dr. Nancy Rayprovided antibody against nucleoside diphosphate kinase, as well as anti-idiotypic antibody against E. coli nucleoside diphosphate kinase. Purified T4 topoisomerase holoenzyme was a gift from Dr. Kenneth Kreuzer (Duke University).Kate McGaughey provided the IAsys biosensor cuvette with immobilized T4 gene 32 protein. TABLE OF CONTENTS Page 1. Background: Protein-protein interactions in DNA precursor biosynthesis 1 Overview of the bacteriophage T4 infectious cycle 1 DNA precursor biosynthesis and replication 2 Bacteriophage T4 dNTP synthetase complex 3 Escherichia coli enzymes with nucleoside diphosphate kinase activity 7 Escherichia coli ndk mutants 8 Novel NDP kinase 12 Other kinases of Escherichia coli which phosphorylate nucleosides or nucleotides 14 Regulation of nucleoside diphosphate kinase genes and proteins 15 Nucleoside diphosphate kinase gene family 17 Other roles of NDP kinase 19 G-proteins and signal transduction 20 NDP kinase interactions with G-proteins 21 Prokaryotic G-protein homologs 22 Thesis aims 23 Experimental approaches 24 Protein-protein interactions involving E. coli kinases in the T4 dNTP synthetase complex and the T4 replication apparatus 24 Basis for nucleotide pool perturbations in an E. coli ndk mutant 28 dNTP synthesis by adenylate kinase: a novel NDP kinase or a novel (dNTP:AMP) phosphotransferase activity? 30 TABLE OF CONTENTS (Continued) Page 2. Materials and Methods 33 Materials 33 Methods 34 3. Phenotypes of Escherichia coli ndk Mutants 55 Effect of ndk disruption on E. coli growth rate and on T4 infection 55 Confirmation that the novel NDP kinase is adenylate kinase 60 4. A novel (dNTP:AMP) phosphotransferase activity of adenylate kinase can complement nucleoside diphosphate kinase deficiency in E. coli 66 Purification of E. coli adenylate kinase 66 Substrate preference of adenylate kinase 69 NDP kinase activity of Adk 69 Novel (dNTP:AMP) phosphotransferase activity of Adk 72 5. Escherichia coli ndk disruption causes increased CTP synthetase activity and expansion of pyrimidine ribonucleotide and deoxyribonucleotide pools 80 Does Adk cause a dCTP pool perturbation in an ndk disruption mutant? 80 No substrate preference for dCDP 80 Adenylate kinase, ribonucleotide reductase and dCTP pool perturbation 82 Detailed nucleotide pool analysis of parental and ndk strains 83 Increased CTP synthetase activity in the ndk strain 88 Model for CTP and dCTP pool elevations in the ndk strain 93 TABLE OF CONTENTS (Continued) Page 6. Interactions between Escherichia coli nucleotide kinases and bacteriophage T4 DNA replication proteins 96 T4 dNTP synthetase complex and T4 Replication Machinery 96 Electrophoretic mobility shift assay