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Single-Molecule Studies of Mechanical and Helicase-Catalyzed Disruption of Nucleic Acid Duplexes

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Single-Molecule Studies of Mechanical and Helicase-Catalyzed Disruption of Nucleic Acid Duplexes © 2017 Kevin D. Whitley SINGLE-MOLECULE STUDIES OF MECHANICAL AND HELICASE-CATALYZED DISRUPTION OF NUCLEIC ACID DUPLEXES BY KEVIN D. WHITLEY DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics and Computational Biology in the Graduate College of the University of Illinois at Urbana-Champaign, 2017 Urbana, Illinois Doctoral Committee: Associate Professor Yann R. Chemla, Chair Professor Oleksii Aksimentiev Professor Paul R. Selvin Professor Taekjip Ha, Johns Hopkins University Abstract Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair. ii To my parents Linda and Scott and my sister Susan iii Acknowledgments I would first like to thank Yann Chemla for being such an excellent advisor over the past six years. His guidance has molded me into the scientist that I am today. Although I came from a biochemistry background with little experience in physics, he taught me how to think like a physicist. Furthermore, his hands-on training in instrumentation helped me numerous times when I was aligning optics. I am always grateful for the time he spends carefully reading and editing my papers, and for the advice he gives during practice presentations. I would also like to thank the members of my prelim/thesis committees for reading my documents and offering helpful suggestions: Prof. Oleksii Aksimentiev, Prof. Sua Myong (Johns Hopkins), Prof. Taekjip Ha (Johns Hopkins), and Prof. Paul Selvin. I also thank Matt Comstock, who was a postdoc in our lab before taking a faculty position at Michigan State University. As a postdoc, he worked closely with me during my early years in the lab to show me how to perform experiments and analyze data. Even after moving to Michigan he was a valuable collaborator on nearly every project on which I worked. Next I would like to thank all my other past and present labmates: Rustem Khafizov, Taejin Lance Min, Markita Landry, Zhi Qi, Patrick Mears, Kiran Girdhar, Sukrit Suksombat, Vishal Kottadiel, Barbara Stekas, Tanya Perlova, Isaac Li, Roshni Bano, Ruopei Feng, Alice Troitskaia, Monika Makurath, and Aniket Ravan. They have been wonderful colleagues and friends who have always been supportive. I will miss our conversations over lunch, nasty afternoon coffee, and colloquium cookies. I also thank Prof. Timothy Lohman (Washington University School of Medicine) and his lab for their collaboration on the work in the latter half of this dissertation. Lohman lab provided us with all the protein I used in Chapters 4, 5, and 6, and our discussions continue to yield insight into these biological systems. I would like to thank Cindy Dodds, the administrative coordinator for the biophysics program, for her help and advice in navigating the requirements of the program. I also extend my thanks to Prof. Bob Clegg, who was the director of the biophysics program when I began. He went out of his way to help me locate a lab during my first year. Without him, I may never have had the opportunity to join Chemla lab. I also learned so much about optics from him by taking his Optical Spectroscopy course and being a TA for his Optical Microscopy course. Next, I thank the Center for the Physics of Living Cells (CPLC) for such a great collaborative environment here in Illinois, and also for providing me with the opportunity to present my research at a conference in Germany (iPoLS 2014). I am also grateful to Susan Flanegin and Sandra Patterson for being such helpful administrators of the Center. iv I also thank the funding agencies that supported my research assistantship. My work was supported by the National Science Foundation (NSF MCB 09-52442 CAREER to Y.R.C.) and the National Institutes of Health (R21 RR025341 and R01 GM120353, both to Y.R.C.). I would also like to thank the members of the Cusanovich lab at the University of Arizona in which I did my undergraduate research: Prof. Michael Cusanovich, Dr. Terry Meyer, Dr. John Kyndt, and John Fitch. This lab was where I first began my research career, and the hands-on experience I received from John Kyndt and John Fitch was invaluable. I also extend my thanks to the members of the Graduate Christian Fellowship (GCF) for being great friends and for fostering such helpful discussions about how to be a Christian that takes both faith and science seriously. I have benefited tremendously from the conversations I have had with my friends in this fellowship that will continue to serve me throughout my career. Finally, I would like to thank my parents and sister for enabling my scientific curiosity throughout my life. I still remember going to the St. Louis Science Center with them many times when I was young and seeing (and being afraid of) the giant animated dinosaurs. My family has always been supportive of my career choice of scientific research, and has continued to support me throughout my seven years of grad school. v Table of Contents 1. Introduction ......................................................................................................... 1 1.1. Nucleic acid elasticity ...........................................................................................................1 1.2. Dynamics of E. coli UvrD helicase .......................................................................................1 1.3. Single-molecule methods to study biomolecules ..................................................................3 1.3.1. Optical tweezers ................................................................................................................................. 3 1.3.2. Fluorescence microscopy ................................................................................................................... 6 1.3.3. Fleezers: fluorescence + optical tweezers .......................................................................................... 9 2. Elasticity of the transition state for oligonucleotide hybridization .............. 11 2.1. Background .........................................................................................................................11 2.1.1. Nucleic acid hybridization in biology ............................................................................................... 11 2.1.2. Hybridization in biotechnology ........................................................................................................ 12 2.1.3. Hybridization in nanotechnology ..................................................................................................... 12 2.1.4. Past studies of hybridization kinetics ............................................................................................... 12 2.2. Measuring the force-dependence of hybridization rate constants and equilibrium free energies.............................................................................................14 2.2.1. DNA constructs and probe design .................................................................................................... 14 2.2.2. Trap + fluorescence assay ................................................................................................................ 14 2.2.3. Obtaining rate constants from fleezers assay ................................................................................... 15 2.2.4. Rate constants show a non-exponential dependence on force .......................................................... 17 2.2.5. Quantification of photobleaching ..................................................................................................... 20 2.2.6. Effect of fluorescent dye on kinetics ................................................................................................. 22 2.3. Modeling the force-dependence of hybridization rate constants and free energies ................................................................................................................24 2.3.1. Rate and equilibrium
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