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DNA Polymerase I INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type o f computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 The Role of Magnesium in Hydrolytic Nuclease Enzymes. The Use of Inert Chromium and Cobalt Probes in Understanding Magnesium Activation. Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University by Christopher B. Black. B.Sc. ******** The Ohio State University 19% Dissertation Committee: Approved by: Dr. Andrew Wojcicki < j A G u ' Dr. Sheldon G. Shore Adviser Dr. James A. Cowan, Adviser Department of Chemistry UMI Number: 9630854 UMI Microform 9630854 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 To Beth, Tess, my Family and Friends, for their support, friendship and love ACKNOWLEDGEMENTS I would first like to thank my adviser, Professor J. A. Cowan for his guidance in exploring the field of inorganic biochemistry. His expertise and help has played an integral role in my interest and ability to carry out this research. I would also like to thank both Matthew Foster and Professor Peter Andersen for help with the various chromium complexes. Especially Matt, for persevering through the hot summer months learning the joys of synthetic inorganic chemistry. I am also grateful for the financial support provided by Armoco in the form of an industrial fellowship. Throughout the road from bright-eyed graduate student to wom-in researcher I have made many invaluable friends. These friends have enriched my experience at Ohio State as well as kept my sanity in check. I would like to thank Mark Hartell for teaching me the habits of a native New Yorker and putting up with me our first year at Ohio State. Also, those responsible for keeping my sanity: Patricia Anderson, Thomas J. Corcoran, Glenn Jordan IV (“Woody”), Sang-Mock Lee, Jarrod Marto, Adolfo Muccillo, Harsh Vinayak, and Martin Votel. A special thanks goes to John Graham for the endless discussions on chemistry, Ireland and all of the other esoteric topics lost in the annals of time; also, to Anshu Agarwal for her friendship, patience toward my ignorance of Indian culture and for all of the support while in the Cowan lab. An even more special thanks goes to Elizabeth Groeber and her daughter Theresa for their caring, understanding, support and love throughout my time here at Ohio State. Finally, I thank my family: Jeannie, Bud, Donna, Rick and David. Their concern, care, help and support has been instrumental in completing my education in chemistry. iv VITA August 23, 1968 Bom, Hammond, Indiana 1990 B.Sc., Chemistry, University of Cincinnati, Ohio 1990-1996 Graduate Teaching Associate, The Ohio State University, Columbus Ohio 1994 Armoco Fellowship, The Ohio State University, Columbus, Ohio MANUSCRIPTS 1. Role of Divalent Cations in the 3',5'-Exonuclease Reaction of DNA Polymerase I. Black, C.B. and Cowan, J.A. (1992) ChemTracts, 4, 393-397. 2. The Coordination Chemistry of Divalent Magnesium Ion and Monovalent Potassium Ion in Biological Systems. Black, C.B., Huang, H.-W. and Cowan, J.A. (1994) Coordn. Chem. Rev. 135/136, 165-202. v 3. Quantitative Evaluation of Electrostatic and Hydrogen Bonding Contributions to Metal Cofactor Binding to Nucleic Acids. Black, C.B. and Cowan, J.A. (1994) J. Am. Chem. Soc., 116, 1174-1178. 4. Magnesium Activation of Ribonuclease H. Evidence for One Catalytic Metal Ion. Black, C.B. and Cowan, J.A. (1994) lnorg. Chem., 33, 5805-5808. 5. Magnesium-Dependent Enzymes in Nucleic Acid Processing Enzymes: The Biological Chemistry of Magnesium. Black, C.B. and Cowan, J.A., Cowan, J.A., Ed. (VCH Publishers, Inc., New York, NY, 1995), pp. 137-158. 6. Magnesium-Dependent Enzymes in General Metabolism: The Biological Chemistry of Magnesium. Black, C.B. and Cowan, J.A., Cowan, J.A., Ed. (VCH Publishers, Inc., New York,NY, 1995), pp. 159-183. RESEARCH EXPERIENCE Major Field: Chemistry Studies in Inorganic Biochemistry, Group Theory, Nonmetal, Organometallic and Cluster Chemistry: Professors Bruce E. Bursten, James A. Cowan, Eugene P. Schram, Sheldon G. Shore, Andrew Wojcicki, and Robin P. Ziebarth Research in: •Synthesis of Pt(IV), Ru(II) and Cr(III) complexes for use as metal ion probes in the ribonuclease H and exonuclease III systems. •Bacterial growth (E. coli) and isolation of proteins (ribonuclease H and exonuclease III). vi •Development of UV kinetic assay which monitors enzyme steady-state kinetics utilizing hypochromicity of nucleic acids. •Measurement of steady-state rate constants for ribonuclease H and exonuclease III using an optical stopped-flow apparatus. •Calorimetric studies on ribonuclease H-nucleic acid interactions with direct measurement of n binding sites, K binding constant and AH of binding. •Attended Inorganic Biochemistry Summer Workshop, University of Georgia, Athens. August, 1992. (Hands on experience with CHARMM, EPR and 2D NMR). PROFESSIONAL ORGANIZATIONS 1991 Dec.- Member of American Chemical Society 1992 Jan.- Member of American Association for the Advancement of Science Table of Contents Dedication ............................................................................................................................ ii Acknowledgements ............................................................................................................ iii Vita ....................................................................................................................................... v List of Tables ..................................................................................................................... xiii List of Figures ................................................................................................................... xvi List of Abbreviations .................................................................................................... xxii Chapter Page I. Overview ....................................................................................................................... 1 1.1 Introduction .................................................................................................... 1 1.2 Physicochemical Properties of Magnesium ................................................ 9 1.3 The Biochemistry of Magnesium ................................................................ 11 1.3.1 Structural Role ..................................................................................... 11 1.3.2 Catalytic Role ....................................................................................... 13 1.4 Research Objective ........................................................................................ 21 viii II. Materials and Methods ........................................................................................... 23 2.1 General Materials ............................................................................................ 23 2.2 Bulk Growth of Escherichia coli .................................................................. 23 2.3 Enzyme Purification ....................................................................................... 24 2.3.1 Purification of E. coli RNase H using pSK58 ........................... 24 2.3.2 Purification of E. coli RNase H using pET 21 b(+) .................. 26 2.3.3 Purification of E. coli Exonuclease III ....................................... 29 2.3.4 Purification of the Klenow Fragment from CJ379 ................... 29 2.4 Synthesis of (A • dT) 2 o Hybrid Substrate ..................................................... 39 2.5 Preparation of Chromium Complexes ......................................................... 43 2.6 Estimation of Extinction Coefficients and Turnover Rates ...................... 53 2.6.1 E. coli Ribonuclease III ...................................................................... 54 2.6.2 E. coli Exonuclease III ....................................................................... 55 2.6.3 E. coli DNA Polymerase I Klenow Fragment ................................ 56 2.7 Steady-State
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