Analysis of the Structural Changes Caused by Positive DNA Supercoiling by- Marita Christine Barth B.S. Bioresource Research Oregon State University, 1998 Submitted to the Division of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Macromolecular Biochemistry and Biophysics at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2007 C 2007 Massachusetts Institute of Technology. All rights reserved. Signature of Author: Division of Biological Engineering January 12, 2007 Certified by: (I Peter C. Dedon Professor of Toxicology and Biological Engineering Thesis Supervisor Accepted by: L". % SLr/ .,: Alan Grodzinsky Irof's r of Biological Engineering rMASSACK·KIEMEWIJf nNte OF TECHNOLOGY /1 Chairman, Co ittee for Graduate Students AUG 0 2 2007I LIBRARIES ARQHNi This doctoral thesis has been examined by a committee of the Division of Biological Engineering as follows: Associate Professor Bevin P. Engelward Chairman Professor Peter C. Dedon Supervisor Professor John M. Essigmann fl,(\ Dr. Richard J. Roberts --- Analysis of the Structural Changes Caused by Positive DNA Supercoiling by Marita Christine Barth Submitted to the Division of Biological Engineering on January 12, 2007 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Macromolecular Biochemistry and Biophysics ABSTRACT The procession of helix-tracking enzymes along a DNA molecule results in the formation of supercoils in the DNA, with positive supercoiling (overwinding) generated ahead of the enzyme, and negative supercoiling (underwinding) in its wake. While the structural and physiological consequences of negative supercoiling have been well studied, technical challenges have prevented extensive examination of positively supercoiled DNA. Studies suggest that at sufficiently high levels of overwinding, DNA relieves strain by adopting an elongated structure, where the bases are positioned extrahelically and the backbones occupy the center of the helix. This transition has only been identified, however, at a degree of supercoiling substantially higher than is generated physiologically. To examine the structural changes resulting from physiological levels of positive DNA supercoiling, I have developed a method for preparing highly purified positively supercoiled plasmid substrates. Based on a method previously developed in this laboratory, this allows for preparation of large quantities of very pure, highly positively supercoiled plasmid. It also expands on earlier methods by exploiting ionic strength to modulate the direction of supercoiling introduced, allowing preparation of either positively or negatively supercoiled substrates. A combination of approaches has been used to elucidate changes to DNA structure that result from physiological levels of positive supercoiling. Enzymatic probes for regions of single-stranded character are not reactive with positively supercoiled plasmid, indicating that stably unpaired regions are not present. Additionally, the effect of supercoiling on the activity of restriction enzymes has been examined. With the enzymes tested, no substantial differences in cleavage rates were observed with either positively or negatively supercoiled substrates. To examine structural changes at a wider range of superhelical densities, design and preparation was undertaken on 2-aminopurine- containing DNA substrates for use in fluorescence studies with a magnetic micromanipulator. Technical limitations rendered these experiments infeasible with current instrumentation, but important insights were gained for future fluorescence-based micromanipulation experiments. A destabilizing effect on the base pairs, however, can be seen using Raman difference spectroscopy, suggesting a subtle shift toward the more extreme extrahelical state. The Raman data suggest that structural adjustments due to positive supercoiling are small but significant, and in addition to the base-pairing effects, alterations are observed in phosphodiester torsion and the minor groove environment, as well as a slight shift in sugar pucker conformation to accommodate lengthening of the DNA backbone. These results point to subtle changes in DNA structure caused by biologically relevant levels of positive superhelical tension and positive supercoiling. All of the changes are consistent with the mechanical effects of helical overwinding and suggest a model in which base pair destabilization in overwound DNA could affect the search mechanisms used by DNA repair enzymes and the binding of other proteins to DNA. Thesis Supervisor: Peter C. Dedon Title: Professor of Toxicology and Biological Engineering Acknowledgements I would like to start by thanking my family, particularly my parents, Merritt and Jenny, my grandmother Mary, my brother Stephen and my aunt Marita Jo Broadus for their support. They never failed to offer encouragement when I needed it, and I couldn't have done this without them. The research experience I gained as an undergraduate was vital to my success in graduate school, and for that I wish to thank my undergraduate thesis advisor, Professor George S. Bailey, as well as Kate Mathews and Dr. Ulrich Harttig from his laboratory. They were all amazingly kind and encouraging, and I appreciate their willingness to work with and trust an undergraduate in a research environment. I have gained a lot from my interactions with all current and former members of the Dedon Lab, and I would like to extend my thanks to all of them. I would particularly like to thank Debra Dederich, who has been invaluable in preparation of substrates for this research, and whose dedication and hard work in the face of many technical challenges is tremendously appreciated. I would also like to thank Dr. C. Eric Elmquist, who performed the MS analyses of chloroacetaldehyde-treated plasmid samples, and Dr. Michael DeMott who assisted greatly with the editing of this thesis. Dr. Koli Taghizadeh, Yelena Margolin, Dr. Min Dong, and Dr. Christiane Collins all gave important input into my research, and have been a pleasure to work with as well. The support staff for the Dedon Lab has been absolutely amazing, and I would like to thank them all. Marcia Weir, Kristine Marzilli and Dawn Erickson have all done much to make my life easier, and have my gratitude. Olga Parkin is in a class all her own, with an uncanny knack for solving any problem that might come up. Her assistance in all matters administrative, as well as her friendship, have meant a great deal to me. I would also like to thank several of the grad students and post-docs whose friendship I have enjoyed through the years: Dr. Maxine Jonas, David Appleyard, Vasileios Dendroulakis, Dr. Can Ozbal, Dr. Teresa Wright, Dr. Joe Newman, Dr. Maryann Timins, Dr. Elaine Chin, Dr. Janice Lansita, Dr. Jane Sohn, and J.P. Cosgrove. This thesis project has involved multiple collaborations, which have significantly enhanced both my education and the quality of the information obtained. I would like to thank Professor George Thomas at the University of Missouri at Kansas City for opening up his lab to me for the Raman spectroscopic studies of supercoiling. Within the Thomas lab, I wish to thank Professor James Benevides for sharing his expertise with me, and allowing me so much instrument time on my visits to Kansas City. Additionally, he and his family were wonderful hosts, and truly made me feel welcome during my stays there. For their collaboration on the 2-aminopurine studies, I wish to thank Professor Peter So and Dr. Serge Pelet for their input and experimental assistance. I would like to thank my thesis committee, Professor Bevin Engelward, Professor John Essigmann and Dr. Richard Roberts for all of their support and input. They've been incredibly encouraging, and have been a wonderful source of challenging questions and intriguing ideas. For help in more ways than I can list, I would like to thank Gavin McNett. His keen editorial eye and assistance with graphics have dramatically improved the quality of this thesis. Beyond that, his kindness, good nature, wit, phenomenal cooking skill, and (above all) patience have been vital to seeing me through the thesis writing process. I find it difficult to adequately express my gratitude and respect. Finally, I would like to thank my thesis advisor, Professor Peter Dedon, for all his support and encouragement through the years. He has been incredibly generous and supportive, and I truly appreciate him sticking with me through what proved to be a very challenging and difficult project. His openness to new ideas and the freedom he has given me to explore different approaches to the problems I have faced have greatly enriched my education. I am truly grateful. Biographical Note Marita Christine Barth was born in 1975 in Dallas, Oregon, and graduated from Dallas High School in 1993. After a summer internship at the Laboratory Services Division of the Oregon Department of Agriculture, she enrolled at Oregon State University in Corvallis, OR. Following a junior year abroad at Lincoln University in Canterbury, New Zealand, she returned to complete a thesis project, entitled "In vitro Mechanisms of Chlorophyllin Anticarcinogenesis Against Dibenzo[a, l]pyrene," in the laboratory of Professor George S. Bailey. She was awarded a Bachelor of Science in Bioresource Research with an option in Toxicology and a minor in Chemistry, in 1998. She subsequently enrolled at MIT, and during the course of her graduate studies has been awarded a National Defense