Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ravindra Bandara Amunugama, B.S. Biophysics Graduate Program The Ohio State University 2011 Dissertation Committee: Richard Fishel PhD, Advisor Jeffrey Parvin MD PhD Charles Bell PhD Michael Poirier PhD Copyright by Ravindra Bandara Amunugama 2011 ABSTRACT Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and following the formation of DNA double strand breaks (DSBs) induced by exogenous stresses such as ionization radiation. HR is also involved in restoring replication when replication forks have stalled or collapsed. Defective recombination machinery leads to chromosomal instability and predisposition to tumorigenesis. However, unregulated HR repair system also leads to similar outcomes. Fortunately, eukaryotes have evolved elegant HR repair machinery with multiple mediators and regulatory inputs that largely ensures an appropriate outcome. A fundamental step in HR is the homology search and strand exchange catalyzed by the RAD51 recombinase. This process requires the formation of a nucleoprotein filament (NPF) on single-strand DNA (ssDNA). In Chapter 2 of this dissertation I describe work on identification of two residues of human RAD51 (HsRAD51) subunit interface, F129 in the Walker A box and H294 of the L2 ssDNA binding region that are essential residues for salt-induced recombinase activity. Mutation of F129 or H294 leads to loss or reduced DNA induced ATPase activity and formation of a non-functional NPF that eliminates recombinase activity. DNA binding studies indicate that these residues may be essential for sensing the ATP nucleotide for a functional NPF formation. ii An intriguing structural motif in the nucleotide-binding subunit interface of RAD51 is known as the ATP cap. The RAD51 family of recombinases contains a conserved aspartate that forms a salt bridge with the terminal phosphate of ATP, which is the likely source of a nonphysiological cation requirement for the formation of an active RAD51 NPF. In Chapter 3, I describe the biochemical analyses of a lysine substitution mutation of this conserved aspartate. The prototypical bacterial recombinase RecA as well as most RAD51 paralogs contains a conserved lysine at the analogous position. The HsRAD51(D316K) substitution mutant possess a reduced protein turnover from DNA that results in improved recombinase functions. Structural analyses indicates that HsRAD51(D316K) and its archaebacterial homolog form extended active NPF without the requirement of salt. These studies suggest that HsRAD51(D316) salt bridge may function as a conformational sensor that enhances RAD51 turnover at the expense of recombinase activity. In Chapter 4, I examination the role of the RAD51 paralog complex RAD51B- RAD51C as a potential mediator of RAD51 catalyzed D-loop formation. Modeled structures indicate that RAD51, RAD51B and RAD51C appear to have similar domain orientations within a NPF. Furthermore, RAD51B-RAD51C form stable complexes on ssDNA and partially stabilizes RAD51 NPF from the anti-recombinogenic activity of BLM. At sub-stoichiometric levels, RAD51B-RAD51C may modestly stimulate RAD51 mediated D-loop formation in presence of RPA. Collectively, these studies provide additional mechanistic and structural insight into the regulation of the RAD51 NPF during the homology search and strand exchange processes that is critical for efficient HR in human cells. iii Dedicated to Parents, my wife and my son iv ACKNOWLEDGMENTS It had always been my passion to study biochemical mechanisms of DNA damage repair and it was no surprise that from the first day of my rotation in Fishel Lab I decided to join and pursue the work that I have done so far. I would like to extend my heartfelt gratitude to my PhD advisor Richard Fishel for his continuous support throughout my graduate education in his lab. I greatly appreciate the freedom he allowed me to have to independently address research problems. Many a times I learned the ropes the hard way. But that gave me confidence to pursue a future career as an independent scientist. The fact that Rick would often challenge my scientific opinions compelled me to be well versed with the literature. I would also like to thank him for carefully reading and editing the drafts of my manuscripts. Finally, I look forward to having him as a mentor for many years ahead for guidance and support. I would like to thank the members of my committee Chuck Bell, Jeffrey Parvin and Michael Poirier for their guidance and advise throughout my graduate career. I thank Steve Kowalczykowski, Lorraine Symington, Wolf-Dietrich Heyer and Steve West for stimulating discussions, advice and opinions on the confusing world of Recombination. I am extremely grateful to Kang-Sup Shim (KS) for advice and teaching me the techniques initially and for many long hours of thoughtful discussions. Also I would like to thank Naduparambil Jacob who has been a wonderful collaborator and a colleague v over the years. I am thankful to Sarah Javaid, Kristine Yoder, Michael Mcilhatton and Samir Acharya for their friendship, advice and helpful discussions. I also like to acknowledge Tom Clanton former director of the Biophysics graduate program, Ralf Bundschuh current co-director and Susan Hauser for their assistance. I am grateful to my parents and my brother for believing in me and all their support during my early education. It was the inspiration that I received from my father, being an academic himself, which made me choose the path of academia. Also, I am thankful to my friends Indi, Sharon, Chamika, Mekhala, Suresh, Harshi, Shekar and Poorani for their friendship and numerous fun times we had that never made us miss home. I am also grateful to my mother-in-law for taking care of our son for a whole year that allowed my wife and me to continue with our education peacefully. Finally, no words can express the how grateful I am to my dear wife Nirodhini for all her love, support and most importantly her patience throughout our busy years. I am also thankful to my dear son Rehan, who brought us an enormous amount of joy, happiness and meaning to our lives. I want you two to know that even though I have had many shortfalls in trying to be the ideal husband and father, you two are the most important people in my life and the motivation that drives me forward. vi VITA 1977................................................................Born- Paris, France 2003................................................................B.S. Honors, Biochemistry and Molecular Biology, University of Colombo, Sri Lanka 2003-2004………………………………….. Teaching Assistant in Biochemistry, Molecular Biology and Chemistry, University of Colombo, Sri Lanka 2004-2005 ......................................................Graduate Teaching Assistant, Department of Chemistry, University of Iowa, USA 2005-Present ..................................................Graduate Research Associate, Department of Molecular Virology, Immunology and Medical genetics, The Ohio State University, USA vii PUBLICATIONS 1. Amunugama R. and Fishel R. (2011) Subunit interface residues F129 and H294 of human RAD51 are essential for recombinase activity. Plos One 6(8), e23071. 2. Charbonneau, N., Amunugama, R., Schmutte, C., Yoder, K., and Fishel, R. (2009) Evidence that hMLH3 functions primarily in meiosis and in hMSH2-hMSH3 mismatch repair. Cancer Biol Ther 8(14), 1421-20. 3. Su, X., Jacob, N. K., Amunugama, R., Hsu, P.-H., Fishel, R., and Freitas, M. A. (2009) Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry. Analytical Biochemistry 388, 47-55. 4. Su, X., Jacob, N. K., Amunugama, R., Lucas, D. M., Knapp, A. R., Ren, C., Davis, M. E., Marcucci, G., Parthun, M. R., Byrd, J. C., Fishel, R., and Freitas, M. A. (2007) Liquid chromatography mass spectrometry profiling of histones. J Chromatogr B Analyt Technol Biomed Life Sci 850, 440-54. 5. Ikura, T., Tashiro, S., Kakino, A., Shima, H., Jacob, N., Amunugama, R., Yoder, K., Izumi, S., Kuraoka, I., Tanaka, K., Kimura, H., Ikura, M., Nishikubo, S., Ito, T., Muto, A., Miyagawa, K., Takeda, S., Fishel, R., Igarashi, K., and Kamiya, K. (2007) DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics. Mol Cell Biol 27, 7028-40. FIELD OF STUDY Major Field: Biophysics viii TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................ v VITA................................................................................................................................. vii LIST OF TABLES .......................................................................................................... xii LIST OF FIGURES ....................................................................................................... xiii CHAPTER 1: Homologous Recombination in Eukaryotes ................................................ 1 1.1 ABSTRACT .................................................................................................................. 2 1.2 INTRODUCTION TO HOMOLOGOUS RECOMBINATIONAL REPAIR ...........