University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2009 Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae James Wesley Fairman University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Fairman, James Wesley, "Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae. " PhD diss., University of Tennessee, 2009. https://trace.tennessee.edu/utk_graddiss/49 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by James Wesley Fairman entitled "Structure- Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Biochemistry and Cellular and Molecular Biology. Chris G. Dealwis, Major Professor We have read this dissertation and recommend its acceptance: David A. Brian, Elias J. Fernandez, Elizabeth E. Howell, Ana A. Kitazono Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by James Wesley Fairman entitled “Structure- Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Biochemistry, Cellular, and Molecular Biology. Chris G. Dealwis, Major Professor _________ We have read this dissertation and recommend its acceptance: David A. Brian____________________ Elias J. Fernandez_________________ Elizabeth E. Howell _______________ Ana A. Kitazono__________________ Accepted for the Council: ______________________________________ Carolyn R. Hodges Vice Provost and Dean of the Graduate School Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville James Wesley Fairman August 2009 ACKNOWLEDGEMENTS I would like to start by thanking my mentor Chris Dealwis for allowing me to join his laboratory and for his guidance during my graduate studies at the University of Tennessee. He is always willing to push my boundaries, both mentally and as a scientist, and I commend him for that. Second, I would like to thank all the remaining members of my committee: Elias Fernandez, Elizabeth Howell, Ana Kitazono, and David Brian. Their input and advice has been critical to the completion of my research, the yeast Rnr project in particular. In addition, I would like to acknowledge our collaborator Julian Simon at the Fred Hutchinson Cancer Research Center, as he provided the basis for half of my dissertation research project. I would also like to thank Yun Yen at the City of Hope for providing me with plasmid containing the cDNA of human Rnr1. All the members of the Dealwis lab have contributed either directly or indirectly to the work presented here, and it would not have been possible without both their friendship and dedication. Tomoaki Uchiki was involved in both my training and the initial stages of my research projects. He also developed the purification scheme for yeast Rnr, a protocol I have performed more times than I can possibly remember. Anna Gardberg, Brad Bennet, and Hai Xu were all heavily involved in my training as an X-ray crystallographer, teaching me the intricacies of various crystallographic refinement and analysis software. Sanath Wijerathna, a fellow graduate student in the Dealwis lab, has been instrumental in providing critical analysis, data, and moral support throughout the years. Structure factor data for the yeast hexamer and size exclusion data provided by Sanath will be instrumental in publishing of the research in Chapter 4 of this dissertation into the scientific literature. Beth Helmbrecht and John Lamaccia are two ii undergraduate students which were both indispensable to the Rnr project. They performed site- directed mutagenesis experiments and purified countless milligrams of recombinant protein during their time in the Dealwis lab. Joe Racca not only discovered the original conditions for the crystallization of the yeast Rnr enzyme, but has also been a continual source of intellectual stimulation, friendship, and mental support for over six years, and hopefully many more to come. My friends and family have been a source of emotional, mental, and even financial support during my time as a graduate student. Mom and dad, thank you for your patience, I told you this day would come eventually! Ross, Matthew, Mark, Foley, and anyone else from the Knoxville crew I haven‟t mentioned, you guys rock! Thank you for forcing me to get out of the lab on the weekends to do something other than science! iii ABSTRACT Sufficient pools of deoxyribonucleotide triphophates (dNTPs) are essential for the high fidelity replication and repair of DNA, the hereditary material for a majority of living organisms. Ribonucleotide reductase (Rnr) catalyzes the rate-limiting step of de-novo DNA synthesis, the reduction of ribonucleosides to deoxyribonucleosides. Since the cell relies primarily upon ribonucleotide reductase for its dNTPs, both the cellular levels and activity of Rnr are heavily regulated, especially when DNA damage occurs or during replication blocks in the cell cycle. If dNTP pools become too high, too low, or imbalanced, genomic instability results, leading to either the formation of cancerous cells or cell death. High levels of dNTPs are required by actively propagating cells for the replication of new DNA molecules. Therefore, Rnr makes an excellent target for anti-cancer, anti-microbial, and anti-fungal chemotherapeutic agents. Deficiencies in the cellular mismatch repair (MMR) machinery have been linked to genetic instability and carcinogenesis. Two alleles of Rnr1 were recently discovered, Rnr1S269P and Rnr1S610F, which have a mismatch repair synthetic lethal (msl) phenotype in Saccharomyces cerevisiae cells with missing or defective MMR genes. To uncover the molecular mechanism of the msl phenotype in these two mutants, recombinant Rnr1p-S269P and Rnr1p-S610F were subjected to in vitro activity assays, X-ray crystallography, and in vitro nucleoside-binding assays (Chapter 3). The Rnr1S269P allele was shown to dysregulate specificity cross talk by X- ray crystallography experiments, leading to reduced levels of dATP in the cell. A 2-fold reduction in binding of ADP substrate was observed in the Rnr1S610F allele, however reduction of the kcat is believed to cause the observed msl phenotype in this mutant. iv The first X-ray crystal structures of the large subunit of ribonucleotide reductase from Homo sapiens (hRRM1) are also presented here (Chapter 4). The hRRM1●TTP and hRRM1●TTP●GDP structures describe the binding of effector and substrate to the specificity and catalytic sites. In addition, the two structures hRRM1●TTP●ATP and hRRM1●TTP●dATP are the first X-ray crystal structures of Rnr from any species with the allosteric activity site occupied with the natural ligands ATP and dATP. Size exclusion chromatography data and a low resolution X-ray crystal structure of hexameric S. cerevisiae Rnr provide a model for dATP- dependent oligomerization. v TABLE OF CONTENTS 1. BACKGROUND AND INTRODUCTION ..................................... 1 1.1 INTRODUCTION AND OVERVIEW ............................................................................. 1 1.2 CLASSIFICATION OF RIBONUCLEOTIDE REDUCTASES ........................................... 2 1.2.1 Class I Ribonucleotide Reductases .................................................................. 3 1.2.2 Class II Ribonucleotide Reductases ................................................................ 9 1.2.3 Class III Ribonucleotide Reductases ............................................................. 10 1.3 CATALYSIS IN CLASS Ia RIBONUCLEOTIDE REDUCTASES ................................... 11 1.3.1 Ribonucleosides to Deoxyribonucleosides .................................................... 11 1.3.2 Catalytic Mechanism of Ribonucleotide Reductases .................................... 14 1.3.3 Catalytic Inhibitors of Class Ia Ribonucleotide Reductases ........................ 17 1.4 REGULATION OF CLASS Ia RIBONUCLEOTIDE REDUCTASES ............................... 21 1.4.1 Allosteric and Oligomeric Regulation ........................................................... 21 1.4.2 Ribonucleotide Reductase and its Regulation in S. cerevisiae ..................... 28 1.4.3 Ribonucleotide Reductase and its Regulation