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Variant Requirements for DNA Repair Proteins in Cancer Cell Lines That Use Variant requirements for DNA repair proteins in cancer cell lines that use alternative lengthening of telomere mechanisms of elongation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alaina Rae Martinez Biomedical Sciences Graduate Program The Ohio State University 2016 Dissertation Committee: Dr. Jeffrey D. Parvin, Advisor Dr. Joanna Groden Dr. Amanda E. Toland Dr. Kay F. Huebner Copyright by Alaina Rae Martinez 2016 Abstract The human genome relies on DNA repair proteins and the telomere to maintain genome stability. Genome instability is recognized as a hallmark of cancer, as is limitless replicative capacity. Cancer cells require telomere maintenance to enable this uncontrolled growth. Most often telomerase is activated, although a subset of human cancers depend on recombination-based mechanisms known as Alternative Lengthening of Telomeres (ALT). ALT depends invariably on recombination and its associated DNA repair proteins to extend telomeres. This study tested the hypothesis that the requirement for those requisite recombination proteins include other types of DNA repair proteins. These functions were tested in ALT cell lines using C-circle abundance as a marker of ALT. The requirement for homologous recombination proteins and other DNA repair proteins varied between ALT cell lines compared. Several proteins essential for homologous recombination were dispensable for C-circle production in some ALT cell lines, while proteins grouped into excision DNA repair processes were required for C- circle production. The MSH2 mismatch repair protein was required for telomere recombination by intertelomeric exchange. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between human cancer cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways. Further studies of all DNA repair pathways in ALT will likely lead to a better understanding of ALT mechanisms and ultimately better ALT-targeted therapeutics. ii Dedication To my grandparents, parents and husband who have worked hard and made sacrifices so that I could have this opportunity and to my siblings for being examples for me to follow. iii Acknowledgments I would like to acknowledge my advisors Dr. Jeffrey Parvin and Dr. Joanna Groden for their guidance and encouragement throughout graduate school. I would like to thank committee members Dr. Amanda Toland and Dr. Kay Huebner for their helpful suggestions, interest in my project and thought-provoking questions. I would like to thank the 9th floor of the BRT; they have been a very collegial and friendly group of cancer researchers. Over the years there have been many members of the Parvin and Groden labs and rotation labs that I have been lucky to have met. Many became not only friends but my “Columbus family” who supported me through the hard times. I am so grateful for those friendships and could not have made it through graduate school without them. I would also like to thank those who have guided me to this point in my life: teachers from Lewiston-Porter; professors from The College of Wooster, who believed in my capabilities; Dr. José Lemos who provided me the same opportunities, as a lab technician, as he did his students and prepared me for grad school; to him, Dr. Jacqueline Abranches and Dr. Jessica Kajfasz, who sparked my interest in pursuing a PhD; Lemos lab friends that lent empathetic ears and encouraged me. I thank my family and friends for support, love and belief in me. Lastly, I would like to thank my husband, Senyo. It was difficult to be away from each other for five years but he encouraged me to finish graduate school. I look forward to spending the rest of my life with him, finally! iv Vita May 2007 .......................................................B.A. Biochemistry & Molecular Biology, The College of Wooster 2010 to present ..............................................PhD Candidate, The Ohio State University Publications Acharya, S., Kaul, Z., Gocha, A.S., Martinez, A.R., Harris, J., Parvin, J.D., and Groden, J. (2014). Association of BLM and BRCA1 during telomere maintenance in ALT cells. PLoS One 9: 1–13. Singh, M., Martinez, A.R., Lee, B.S. (2013). HuR inhibits apoptosis by amplifying Akt signaling through a positive feedback loop. J Cell Physio 228: 182-189. Abranches, J., Miller, J.H., Martinez, A.R., Simpson-Haidaris, P.J., Burne, R.A., Lemos, J.A. (2011). The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells. Infect and Immun 79: 2277-2284. Martinez, A.R., Abranches, J., Kajfasz, J.K., Lemos, J.A. (2010). Characterization of the S. sobrinus acid-stress response by interspecies microarrays and proteomics. Mol Oral Micro 25: 331-342. Kajfasz, J.K., Rivera-Ramos, I., Abranches, J., Martinez, A.R., Rosalen, P.L., Derr, A.M., Quivey R.G., Lemos, J.A. (2010). Global regulation by two Spx proteins modulate stress tolerance, survival, and virulence in S. mutans. J Bacteriol 192: 2546-2556. Abranches, J., Martinez, A.R., Kajfasz, J.K., Chávez, V., Garsin, D.A., Lemos, J.A. (2009). The molecular alarmone (p)ppGpp mediates stress responses, Vancomycin tolerance, and virulence in Enterococcus faecalis. J Bacteriol 191: 2248-2256. Kajfasz, J.K., Martinez, A.R., Rivera-Ramos, I., Abranches, J., Koo, H., Quivey, Jr., R.G., Lemos, J.A. (2009). Role of Clp proteins in the expression of virulence properties of Streptococcus mutans. J Bacteriol 191: 2060-2068. Fields of Study Major Field: Biomedical Sciences v Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v Publications ......................................................................................................................... v List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Chapter 1: Functions of DNA Repair Proteins in Alternative Lengthening of Telomere Mechanisms ........................................................................................................................ 1 I. Telomere maintenance ................................................................................................. 2 I.1 Telomere structure and function ............................................................................ 2 I.2 Telomerase ............................................................................................................. 4 I.3 Alternative lengthening of telomeres ..................................................................... 4 II. DNA repair proteins in ALT ...................................................................................... 9 II.1 DNA damage sensors .......................................................................................... 10 II.2 Chromatin modifiers ........................................................................................... 12 vi II.3 Double-strand break proteins .............................................................................. 13 II.4 RecQ-like helicases ............................................................................................. 18 II.5 Excision repair .................................................................................................... 19 Chapter 2: Thesis Rationale and Research Objectives ..................................................... 26 Chapter 3: Differential Requirements for DNA Repair Proteins in Cell Lines Using Alternative Lengthening of Telomere Mechanisms ......................................................... 29 I. Introduction ................................................................................................................ 29 II. Materials and methods .............................................................................................. 32 Cell lines .................................................................................................................... 32 siRNA knockdown ...................................................................................................... 32 Western blots ............................................................................................................. 33 qRT PCR assays......................................................................................................... 33 C-circle assays ........................................................................................................... 33 Telomere sister chromatid exchange assays ............................................................. 34 Homology-directed repair assays .............................................................................. 35 III. Results ....................................................................................................................
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