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Uni International 300 N. Zeeb Road Ann Arbor, Ml 48106 8318416 Pantaleone, David Peter STUDIES ON THE PURIFICATION, CHARACTERIZATION AND MECHANISM OF POLY(ADP-RIBOSE) POLYMERASE FROM CALF THYMUS The Ohio Slate University Ph.D. 1983 University Microfilms International300 N. Zeeb Road, Ann Arbor, M I 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages_______ 2. Colored illustrations, paper or print______ 3. Photographs with dark background______ 4. Illustrations are poor copy_______ 5. Pages with black marks, not original copy____ 6. Print shows through as there is text on both sides of page_______ 7. Indistinct, broken or small print on several pages 8. Print exceeds margin requirements ______ 9. Tightly bound copy with print lost in spine_______ 10. Computer printout pages with indistinct print______ 11. Page(s)_____________lacking when material received, and not available from school or author. 12. Page(s)_____________seem to be missing in numbering only as text follows. 13. Two pages num bered_____________. Text follows. 14. Curling and wrinkled pages_______ 15. Other_______________________________________________________ University Microfilms International STUDIES ON THE PURIFICATION, CHARACTERIZATION AND MECHANISM OF POLY(ADP-RIBOSE) POLYMERASE FROM CALF THYMUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By David P. Pantaleone, B.A. * * * * The Ohio State University 1983 Reading Committee: Approved By Robert M. Mayer Reynaldo C. Pless Ming-Daw Tsai Adviser (/ Department of Chemistry DEDICATION To my wife, Jennifer ii ACKNOWLEDGMENTS I would like to express my gratitude to my adviser, Dr. Robert M. Mayer, for his guidance throughout this research project and the preparation of this dissertation. I also wish to thank the colleagues in my laboratory for their support throughout my endeavors, as well as my family for their encouragement. I would like to thank Mr. Peter Pantaleone and Mr. James Pantaleone for obtaining the fresh calf thymus; also Mr. Herman Guiney for making, with Mr. Peter Pantaleone, the sieving device which aided in the preparation of the DNA-agarose affinity column. Finally, I wish to acknowledge the financial support given me by the Chemistry Department. iii VITA June 17, 1955 ....... Born - Joliet, Illinois 1977 ................. B.A., Lewis University Romeoville, Illinois 1977-1982 ............ Teaching Assistant Chemistry Department The Ohio State University Columbus, Ohio 19 82-1983 ............ Research Associate Chemistry Department The Ohio State University Columbus, Ohio FIELD OF STUDY Major Field: Biochemistry TABLE OF CONTENTS Page DEDICATION ................................................ ii ACKNOWLEDGMENTS ............. iii VITA .......................... iv LIST OF TABLES ........................................... ix LIST OF FIGURES .................................................................................................................................. X Chapter I. INTRODUCTION ............. 1 A. Enzyme Reaction ............................. 2 1. Reactants a. Donor Substrate b. Acceptor Substrates 2. Product a. Structure of Poly(ADP-ribose) b. Poly(ADP-ribose) Degradation 3. Mechanism of Catalysis 4. Purification and Properties of Poly(ADP-ribose) Polymerase a. Poly(ADP-ribose) Synthesis In Vitro as Influenced by Histones B. Physiological Role of Poly(ADP-ribose) ... 37 1. DNA Repair 2. Differentiation and Poly(ADP-ribose) 3. DNA Synthesis C. Purpose of this Investigation ............. 48 v CONTENTS (continued) Page II. MATERIALS AND METHODS ............................ 50 A. Materials ............ 50 1. Tissue 2. Enzymes and Proteins 3. Nucleotides and Related Compounds 4. Chromatography Materials a. Preparation and Assay of DNA- agarose 5. Filtration Membranes 6. Chemicals 7. Synthesis of ADP-ribonolactone B. Methods ........................................ 65 1. Assay Procedure a. Millipore Filter Assay b. Paper Chromatography Assay 2. Radioactive Analysis a. Aqueous Samples b. Polyacrylamide Gel Slices 3. Chromatographic and Electrophoretic Methods a. Paper Chromatography b. Column Chromatography i. General Equilibration Techniques ii. General Monitoring c. Paper Electrophoresis d. Thin Layer Electrophoresis (TLE) e. SDS-Polyacrylamide Gel Electro­ phoresis i. Electrophoresis According to Weber and Osborn (0.1% SDS) ii. Electrophoresis According to Fairbanks et al. (1.0% SDS) f. Acid-Urea Polyacrylamide Gel Electro­ phoresis g. Isoelectric Focusing VI CONTENTS (continued) Page 4. Preparation of Poly(ADP-ribosyl)ated Polymerase a. SDS-PAGE of Poly(ADP-ribosyl)ated Material b. Average Chain Length Analysis 5. Protein Assay 6. General Determinations III. RESULTS ............................................ 76 A. Purification of Poly(ADP-ribose) Polymerase from Calf Thymus ................ 77 1. Crude Extract 2. Ammonium Sulfate Fractionation 3. DNA-agarose Column Chromatography 4. Hydroxylapatite Column Chromatography 5. Blue Sepharose CL-6B Column Chromato­ graphy 6. Separation on the Basis of Molecular Weight a. Concentration on Hydroxylapatite b. Sephadex G-150 Column Chromatography 7. Summary B. Homogeneity ................................... 96 1. Polyacrylamide Gel Electrophoresis in the Presence of SDS 2. Isoelectric Focusing 3. Qualitative Product Evaluation C. Properties of Poly(ADP-ribose) Polymerase .................................... 114 1. Requirements for Maximal Activity 2. Storage of the Purified Enzyme 3. Stability of the Purified Enzyme 4. Molecular Weight Determination vii CONTENTS (continued) Page a. SDS—PAGE According to Weber and Osborn b. SDS-PAGE According to Fairbanks et a l . Product Analysis D. Mechanistic Studies 139 1. Isotope Exchange Reaction 2 . Binding Sites a. Substrate Site i. Kinetic Studies with Nico­ tinamide ii. Substrate Derivatives b. Acceptor Site i. DNA and Whole Histone in Equal Proportions ii. Whole Histone and Histone HI versus DNA Concentration iii. Effect of NAD+ Concentration and Histone HI on ADP-ribose Incorporation c. Automodification Site i. Effect of Acceptor Histone HI on the Automodification Reac­ tion. Average Chain Length A n a l y s i s . IV. DISCUSSION 177 V. BIBLIOGRAPHY 193 viii LIST OF TABLES Table Page 1. Tissues and Cells from which Poly(ADP-ribose) has been Purified............................... 28 2. Properties of Poly(ADP-ribose) Polymerase Purified from Calf Thymus by Ito et a l . (68).......................................... 30 3. Electrophoretic Analysis of Nucleotides Along with ADP-ribonolactone................... 64 4. Purification of Poly(ADP-ribose) Polymerase 95 5. Requirements of Purified Enzyme for Poly (ADP-ribose) Synthesis..................... 115 6. Stability of Poly(ADP-ribose) Polymerase Frozen in Liquid Nitrogen versus Bulk Frozen as a Function of Time at -70°C........ 122 7. Effect of Substrate Analogs on Poly (ADP-ribose) Synthesis..................... 151 8. Effect of Kistone Hi on the Automodification Reaction.......................................... 176 9. Properties of Poly(ADP-ribose) Polymerase from Calf Thymus Purified as Described in This Study .................................... 179 ix LIST OF FIGURES Figure Page 1. Structure of the Substrate, NAD+ ................... 5 2. A. Linear Structure of Poly(ADP-ribose) B. Repeating Unit of Poly (ADP-ribose) ...........