Fluorescence Quenching of A

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 of 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 IJMI 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 Zed) Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 ROLE OF HEXAMERIC STRUCTURE AND dTTPase ACTIVITY IN BACTERIOPHAGE T7 HELICASE-CATALYZED UNWINDING OF DOUBLE STRANDED DNA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philoshophy in the Graduate School of The Ohio State University By Manju M. Hingorani, B.Pharm. ***** The Ohio State University 1996 Dissertation Commitee: Approved by Smita S. Patel Edward J . Behrman Alan M. Lambowitz Adviser Richard P. Swenson Department of Biochemistry UMI Number: 9630900 UMI Microform 9630900 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 my family ii ACKNOWLEDGEMENTS Thank you Smita Patel for your guidance and encouragement. And of course many thanks to you my colleagues and friends, Todd Washington, Kristen Moore, Yiping Jia, Peter Ahnert, Amar Kumar, and Anish Konkar, for the marvelous discussions and cat-fights; I will miss them. VITA November 14, 1968....................... Born - Bombay, India 1990.... ................................. B. Pharm. Bombay University, India 1991-1993 ................................ Teaching assistant, The Ohio State University 1993-present............................. Reasearch assistant, The Ohio State University PUBLICATIONS Hingorani, M. M. & Patel, S.S. (1996) Cooperative Interactions of Nucleotide Ligands are Linked to Oligomerization and DNA Binding in Bacteriophage T7 Gene 4 Helicases, Biochemistry 35f 2218-2228. Egelman, E.H., Xiong, Y., Wild, R., Hingorani, M. M. & Patel, S.S. (1995) Bacteriophage T7 Helicase/Primase Proteins Form Rings Around Single-Stranded DNA that Suggest a General Structure for Hexameric Helicases, Proc.Natl. Acad. Sci. USA 92, 3869-3873. Patel, S. S. & Hingorani, M. M. (1995) Nucleotide Binding Studies of Bacteriophage T7 DNA Helicase-Primase Protein, Biophysical Journal 68, 186s-190s. Patel, S.S., Hingorani, M.M. & Ng, W.M. (1994) The K318A Mutant of Bacteriophage T7 DNA Primase-Helicase Protein is Deficient in Helicase but Not Primase Activity and Inhibits Primase-Helicase Protein Wild-Type Activities by Heterooligomer Formation, Biochemistry 33, 7857-7868. Hingorani, M. M. & Patel, S. S. (1993) Interactions of Bacteriophage T7 DNA Primase/Helicase Protein with Single- Stranded and Double-Stranded DNAs, Biochemistry 32, 12478- 12487. Patel, S. S. & Hingorani, M.M. (1993) Oligomeric Structure of Bacteriophage T7 DNA Primase/Helicase Proteins, J. Biol. Chem. 268, 10668-10675. FIELDS OF STUDY Major Field: Biochemistry Enzymology of a nucleic acid binding protein including structural analysis and mechanistic studies using steady- state and transient-state kinetics. TABLE OF CONTENTS DEDICATION.............................................. ii ACKNOWLEDGEMENTS....................................... iii VITA.................................................... iv LIST OF TABLES......................................... ix LIST OF FIGURES........................................ x LIST OF SCHEMES........................................ xiii CHAPTER PAGE I. INTRODUCTION AND LITERATURE REVIEW............... 1 A. INTRODUCTION TO DNA HELICASES........... 1 B. BACTERIOPHAGE T7 GENE 4 HELICASES...... 9 C. RATIONALE AND AIM OF Ph.D. DISSERTATION RESEARCH..................................... 12 II. OLIGOMERIC STRUCTURE OF BACTERIOPHAGE T7 HELICASES......................................... 14 Abstract..................................... 14 Introduction................................ 15 Materials and Methods....................... 17 Results...................................... 22 Discussion................................... 48 III. DNA BINDING PROPERTIES OF BACTERIOPHAGE T7 HELICASES......................................... 55 Abstract..................................... 55 Introduction................................ 56 Materials and Methods....................... 58 Results...................................... 74 Discussion................................... 132 vii IV. INTERACTIONS OF BACTERIOPHAGE T7 HELICASES WITH NUCLEOTIDE LIGANDS................... 144 A. CHARACTERIZATION OF THE NUCLEOTIDE BINDING SITE................................ 144 Abstract.................................... 144 Introduction................................ 145 Materials and Methods....................... 147 Results...................................... 157 Discussion.................................. 190 B. COOPERATIVE NUCLEOTIDE BINDING EFFECTS HEXAMER FORMATION AND DNA BINDING.......... 199 Abstract..................................... 199 Introduction................................ 200 Materials and Methods....................... 203 Results...................................... 215 Discussion.................................. 247 V. KINETIC MECHANISM OF HELICASE-CATALYZED dTTP HYDROLYSIS.................................. 259 Abstract..................................... 259 Introduction................................ 261 Materials and Methods....................... 263 Results...................................... 267 Discussion.................................. 280 APPENDICES............................................. 296 A. Abbreviations................................. 296 B. Buffers....................................... 300 C. Oligodeoxynucleotide sequences............... 303 D. Chemical structures.......................... 304 LIST OF REFERENCES.................................... 305 viii LIST OF TABLES 1. Equilibrium constants for ssDNA binding to 4A' protein.................................... 98 2. Steady-state kinetic parameters of M13 ssDNA-stimulated dTTPase activity of 4A' and 4A'/K318A proteins.................... 159 3 . Stoichiometry, Kd, and Hill coefficient values for dTMP-PCP binding to 4A' protein.............. 228 4. Parameters of pre-steady-state acid-quench kinetics of dTTP hydrolysis.......... 272 5. Parameters of pre-steady-state pulse-chase kinetics of dTTP hydrolysis....................... 279 ix LIST OF FIGURES 1. Purity of 4A' protein analyzed on SDS-PAGE....... 24 2. Nondenaturing polyacrylamide gel electrophoresis of 4A'............................ 25 3. Standard plot of molecular size markers for HPLC gel-filtration........................... 27 4. Gel-filtration chromatography of 4A' in the absence of nucleotide ligands............. 29 5. High pressure gel-filtration chromatography of 4A' and 4B in the presence of nucleotide ligands................................ 32 6. High pressure gel-filtration chromatography of 4A' and 4B in the presence of MgdTMP-PCP and ssDNA.............................. 38 7. Negative-staining electron microscopy of T7 gene 4 proteins............................. 42 8. Image analysis of 4A' and 4B protein rings....... 47 9. Possible modes of interaction between the hexameric ring and ssDNA...................... 53 10. A sample dot-blot of the nitrocellulose-DEAE membrane binding assay............................ 7 5 11. Single-stranded DNA binding to increasing concentrations of T7 gene 4 proteins............. 78 12. Fluorescence-based assay for DNA binding to 4A'.. 85 13. Native polyacrylamide gel electrophoresis of 4A'.............................................. 88 14. Titration of 4A' and 4B with increasing concentrations of ssDNA........................... 95 15. Nuclease protection of poly(dT) by 4A' and 4B proteins............................... 101 x 16. Duplex DNA binding to increasing concentrations of 4A' protein..................................... 106 17. Competitive binding of ssDNA and dsDNA to increasing concentrations of 4A' protein......... 110 18. Competitive binding of ssDNA and dsDNA at constant 4A' concentrations................... 113 19. Cross-linking of 4A' hexamers around circular M13 ssDNA and linear ssDNA........................ 118 20. Electron micrographs of 4A' and 4B proteins assembled on circular M13 ssDNA.................. 122 21. Polarity of 4B rings on ssDNA..................... 124 22. Three-dimensional reconstruction of the 4B protein ring...................................

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