The roles of hMSH4-hMSH5 and hMLH1-hMLH3 in meiotic double strand break repair DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Randal James Soukup Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2016 Dissertation Committee: Richard Fishel, PhD, Advisor Mark Parthun, PhD Charles Bell, PhD Mamuka Kvaratskhelia, PhD Copyrighted by Randal James Soukup 2016 Abstract The DNA double strand break is a highly cytotoxic DNA lesion. Mouse and human mitotically dividing cells experience ~10 double strand breaks (DSBs) per day that are often repaired through non-homologous end joining and result in the accumulation of short insertions and deletions. However, in prophase I of meiosis, ~400 double strand breaks are introduced into primary mouse spermatocytes by the endonuclease SPO11. The cell undergoes a cell-wide DSB repair response which functions to repair each break, and in doing so, pair homologous chromosomes for segregation at the outset of meiosis I. This process generates genetic crossovers between the homologous chromosomes, which are required for accurate chromosome segregation and are also the basis for the reshuffling of genes between maternal and paternal chromosomes. At the center of this DNA repair process is the Holliday Junction, which physically links homologous chromosomes and whose resolution defines the outcome to a genetic crossover or gene conversion event. A number of proteins involved in mitotic DSB repair are also involved with the meiotic process. However, MSH4-MSH5 and MLH1-MLH3 proteins appear to have unique roles in establishing homologous chromosome pairing and segregation during meiotic DSB repair, but do not play any role in mitotic DSB repair. ii Here we used purified hMSH4-hMSH5 to conduct a series of binding experiments with numerous Holliday Junction constructs. We demonstrate binding of mobile, as well as immobile, Holliday Junctions by hMSH4-hMSH5, and the ability to retain ATP bound hydrolysis-independent sliding clamps on a blocked-end mobile Holliday Junction. In addition, we show that the binding of hMSH4-hMSH5 does not appear to distinguish between the stacked-X or planar conformations of the Holliday Junction. The rate of bulk branch migration by an assembled Holliday Junction did not appear to be affected by the addition of hMSH4-hMSH5. The development of a single molecule approach is reported that will ultimately be used to determine whether the protein transiently or kinetically influences branch migration of individual Holliday Junctions. With no protein currently identified that functions to maintain homologous chromosome pairing through segregation or perform the required Holliday Junction resolution prior to segregation, we partially purified and examined the hMLH1-hMLH3 heterodimer that has been shown to be associated with the development of homologous chromosome linkages. Our preparation of hMLH1-hMLH3 does not appear to display any endonuclease activity or stable complex formation with hMSH4-hMSH5. As has been previously reported we do observe an aggregate that appears to form between hMLH1-hMLH3 and Holliday Junctions at very low ionic strengths. Further hMLH1- hMLH3 purification is required for more complex studies to be performed. iii To my wife, Jenna Karras, you are my biggest champion and best friend; I couldn’t have done this without you. To my Dad, for unwavering love, support, guidance, and friendship. To my Mom, for your constant love and encouragement, I will always love and miss you. iv Acknowledgments I would like to thank my advisor, Rick Fishel, for his guidance, support, and mentorship. I thank him for introducing me to the wonders and frustrations of scientific research and for guiding me through the world of Biophysics. I thank the members of my dissertation committee: Dr. Mark Parthun, Dr. Charles Bell and Dr. Mamuka Kvaratskhelia for generously offering their time, support and guidance throughout my graduate career. Your discussion and ideas have been incredibly helpful. I wish to thank Dr. Kristine Yoder for her support, encouragement and kindness. Thank you for keeping your office door open and encouraging my progression to the end. Finally, I want to express my sincere gratitude to past and present members of Dr. Fishel’s and Dr. Yoder’s labs. I thank Nathan Jones, Brooke Britton, Miguel Lopez and Dr. Juana Martin Lopez for their support and friendship. I especially thank Dr. Gayan Senavirathne for his support and scientific discussions, and Dr. Jeungphill Hanne for his commitment to helping and guiding me through single molecule experiments. It has been a pleasure to work with and learn from everyone. v Vita April 15, 1987 ................................................Born – Highland Heights, Ohio May 2005 .......................................................Gilmour Academy May 2009…………………………………….B.S. Biochemistry, Miami University Oxford, Ohio September 2010 to present ............................Graduate Research Associate, The Ohio State University, Columbus, Ohio Publications 1. Xu B, Soukup RJ, Jones CJ, Fishel R, Wozniak DJ. Pseudomonas aeruginosa AmrZ binds to four sites in the algD promoter, inducing DNA-AmrZ complex formation and transcriptional activation. J Bacteriol. 2016 May 16. 2. Xu B, Ju Y, Soukup RJ, Ramsey DM, Fishel R, Wysocki VH, Wozniak DJ. The Pseudomonas aeruginosa AmrZ C-terminal domain mediates tetramerization and is required for its activator and repressor functions. Environ Microbiol Rep. 2016 Feb;8(1):85-90. doi: 10.1111/1758-2229.12354. Epub 2015 Dec 21. 3. Honda M, Okuno Y, Hengel SR, Martín-López JV, Cook CP, Amunugama R, Soukup RJ, Subramanyam S, Fishel R, Spies M. Mismatch repair protein hMSH2- hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate. Proc Natl Acad Sci U S A. 2014 Jan 21;111(3):E316-25. doi: 10.1073/pnas.1312988111. PMID:24395779 Fields of Study Major Field: Molecular, Cellular and Developmental Biology vi Table of Contents Abstract .............................................................................................................................. ii Acknowledgments ............................................................................................................. v Vita .................................................................................................................................... vi List of Tables ..................................................................................................................... x List of Figures ................................................................................................................... xi Chapter 1: Introduction .................................................................................................. 1 1.1 Double strand break repair………………………………………………...1 1.2 Double strand break repair in meiotic cells……………………………….6 1.3 Holliday Junction dynamics and structure…………………………….…..9 1.4 MSH4-MSH5: A MutS homolog involved in meiotic homologous recombination…….………………………………………...12 1.5 hMLH1-hMLH3: A MutL homolog with a role in meiotic recombination………………………………………………...14 Chapter 2: Investigating the mechanism of hMSH4-hMSH5 search for the Holliday Junction branch point and its impact on branch migration.....17 2.1 Introduction……………………………………………………………..17 2.2 Materials and methods…………………………………………………..19 vii 2.3 Results………………………………………………………………...…27 2.3.1 hMSH4-hMSH5 can be purified to near homogeneity…………..27 2.3.2 hMSH4-hMSH5 recognizes immobile Holliday Junctions and form a sliding clamp upon ATP binding……….……………29 2.3.3 hMSH4-hMSH5 also recognizes mobile Holliday Junctions and forms a sliding clamp upon ATP binding………….………..31 2.3.4 hMSH4-hMSH5 shows stronger binding affinity to a mobile Holliday Junction containing a dsDNA tail……………………...33 2.3.5 The Holliday Junction conformation does not appear to impact hMSH4-hMSH5 binding …………………………………….…..35 2.3.6 An hMSH4-hMSH5 sliding clamp can be retained on mobile Holliday Junctions when DNA ends are blocked………….…….36 2.3.7 hMSH4-hMSH5 sliding clamp is retained on tailed mobile Holliday Junctions ……………………………………..………..38 2.3.8 Development of FRET based assembled Holliday Junction branch migration assay………………………………………..................40 2.3.9 Validating the migration rates of our assembled Holliday Junction……………………………………………………….….44 2.3.10 The addition of hMSH4-hMSH5 to assembled Holliday Junctions does not appear to alter the rate of branch migration……..……..45 2.3.11 Pitfalls of assembled Holliday Junction branch migration……….48 2.3.12 Developing a model single molecule branch migratable Holliday Junction………………………………………………...48 2.4 Discussion………………………………………………………………..53 Chapter 3: Determining the in vitro activity of purified hMLH1-hMLH3………….56 3.1 Introduction………………………………………………………………56 3.2 Materials and methods…………………………………………………...58 viii 3.3 Results……………………………………………………………………62 3.3.1 Purification of hMLH1-hMLH3…………………………………62 3.3.2 Testing purified hMLH1-hMLH3 for Endonuclease Activity…………………………………………………………..65 3.3.3 Determination of the DNA substrates which associate with hMLH1-hMLH3…………………………………………………66 3.3.4 hMLH1-hMLH3 recruitment to HJ by hMSH4-hMSH5………...69 3.4 Discussion………………………………………………………………..71 Chapter 4: Discussion………………………………………………………………….73 4.1 Introduction……………………………………………………………...73 4.2 Interaction between hMSH4-hMSH5 and the Holliday Junction………..75