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Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights Into Disease and Function: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2016-07-15 Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation Caroline M. Duffy University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Biochemistry Commons, Molecular Biology Commons, and the Structural Biology Commons Repository Citation Duffy CM. (2016). Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M22P45. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/844 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. STRUCTURAL MECHANISMS OF THE SLIDING CLAMP AND SLIDING CLAMP LOADER: INSIGHTS INTO DISEASE AND FUNCTION A Dissertation Presented By CAROLINE MARY DUFFY Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY July 15, 2016 Biochemistry and Molecular Pharmacology STRUCTURAL MECHANISMS OF THE SLIDING CLAMP AND SLIDING CLAMP LOADER: INSIGHTS INTO DISEASE AND FUNCTION A Dissertation Presented By CAROLINE MARY DUFFY This work was undertaken in the Graduate School of Biomedical Sciences Biochemistry and Molecular Pharmacology The signature of the Thesis Advisor signifies validation of Dissertation content Brian A. Kelch, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Daniel Bolon, Ph.D., Member of Committee Kendall Knight, Ph.D., Member of Committee Nicholas Rhind, Ph.D., Member of Committee Peter Chien, Ph.D., External Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee William Royer, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences July 15, 2016 iii ACKNOWLEDGMENTS It’s truly been a privilege working in Dr. Brian Kelch’s lab. I greatly admire his tenacity, his insatiable love for all science, and his well-documented inability to let a chalk talk go uninterrupted with insightful questions. I appreciate his forthright opinions, support, and deep commitment to his students’ success. Most of all, I will always be grateful he took me into his lab. Despite the challenges grad school threw at me, I feel incredibly lucky to have trained with him and I fully expect he will do great things. I’m thankful to say I’ve genuinely enjoyed working with my lab mates. I could not have done the bulk of my thesis work without the help of Dr. Brendan Hilbert. Not only do I appreciate our friendship, but I admire his meticulous scientific mind. He has made me a better scientist and I’m indebted to him for his expertise, especially for help with all things crystallographic and ITC. Nicholas Stone and Janelle Hayes joined the lab not long after I did and I’m grateful we have been close both in and out of lab. Our small group has always been incredibly supportive of one another and I will miss working with all of them. Working in the Biochemistry and Molecular Pharmacology Department has also been a privilege. I’ve loved having the opportunity to chat science in as many offices as I have over the years, and I have enjoyed the department’s collaborative atmosphere. I appreciate the feedback I’ve received from my awesome TRAC/DEC committee members, past and present: Dr. Bill Royer (my iv fantastic chair), Dr. Sean Ryder, Dr. Nick Rhind, Dr. Ken Knight, Dr. Dan Bolon, and my external member, Dr. Peter Chien. In addition, I received invaluable training, technical support, and/or advice from Dr. Mary Munson, Luca Leone, Ellen Nalivaika, Dr. Djade Soumana, Melissa Greven, Livio Dukaj, Dr. Maggie Heider, Dr. Dave Mofford, and Josh Gardner. It takes a village! I’ve made some life-long friends in grad school, and whether we were commiserating or celebrating, it was always great to have people who knew exactly the frustration and joy you can (sometimes simultaneously) have while working towards your PhD. To my immediate family, Mom, Dad, Meg, and Tim, you have always encouraged me blindly and loudly, and most times I just needed to hear that I could do this. Finally, to my partner-in-crime, Todd Monroe: You have supported me since going to graduate school was a mere thought. Thank you for patiently accepting without complaint the crazy schedule that working in a lab brings, understanding phrases like “running a gel” meant I would probably be late, and “concentrating protein” definitely meant I would be late. For better or worse, many of our relationship milestones coincided with graduate school milestones— I’m sorry again for going to the synchrotron the weekend you were going to propose. I didn’t know. But I got a structure and a husband out of it regardless. Thank you for your unwavering love and patience, your constant support, and thank you for doing most of the dishes and laundry during the time it took to write this thesis. I could not have done this without you. v ABSTRACT Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA. The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partner- binding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding. Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader vi binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery. vii TABLE OF CONTENTS Acknowledgments iii Abstract v Table of Contents vii List of Tables x List of Figures xi List of Third Party Copyrighted Material xiv Preface xv CHAPTER I: INTRODUCTION DNA replication and repair: 2 Replication initiation 5 The replisome 7 DNA damage and repair 9 The sliding clamp 12 Sliding clamps are essential for processive DNA replication 12 Sliding clamps share a widely conserved structure 12 The IDCL is a conserved binding site in all sliding clamps 17 PCNA interacting partners contain conserved binding motifs 20 Additional PCNA roles beyond processivity 22 PCNA in DNA repair 23 Cell cycle and apoptosis 24 Epigenetic inheritance, chromatin assembly and remodeling 25 Open questions and scope of thesis 25 The sliding clamp loader 27 Clamp loader architecture and subunit composition 29 The clamp loader reaction 31 ATP binding 33 Clamp binding 33 Primer/template binding 35 ATP hydrolysis and clamp loader ejection 37 Open questions and Scope of thesis 37 CHAPTER II: A disease causing variant in PCNA disrupts a promiscuous protein binding site Introduction 40 Materials and Methods 45 Results 49 Structure of hPCNA-S228I 49 S228I mutation disrupts PIP binding 56 viii p21CIP PIP-box binding reverts IDCL conformation to that seen for wild-type 63 FEN1 PIP-box binding induces a novel conformation of the IDCL 70 Discussion 74 Implications for PCNA conformation, dynamics and PIP binding 74 Implications for disease 86 S228I CHAPTER III: Investigating the cellular effects of PCNA in a fission yeast model system Introduction 90 A subset of PCNA interactions in DNA repair 91 Rationale for a yeast model system for ATLD2 95 Materials and Methods 98 Results 103 pcn1-S228I is viable and not temperature sensitive 103 pcn1-S228I displays a subtle phenotype in response to select DNA damaging agents 108 Discussion 112 CHAPTER IV: Mechanisms for primer/template binding and ATP hydrolysis in the DNA sliding clamp loader Introduction 117 The sliding clamp loader is required for DNA replication 117 How does the clamp loader engage at primer/template junctions? 121 How does ATP hydrolysis occur around the clamp loader
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