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Investigation of the Homologs Rad51 and Dmc1 Role in Cell Division and Homologous Recombination

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Investigation of the Homologs Rad51 and Dmc1 Role in Cell Division and Homologous Recombination BearWorks MSU Graduate Theses Spring 2018 Investigation of the Homologs Rad51 and Dmc1 Role in Cell Division and Homologous Recombination Amaal Abulibdeh Missouri State University, [email protected] As with any intellectual project, the content and views expressed in this thesis may be considered objectionable by some readers. However, this student-scholar’s work has been judged to have academic value by the student’s thesis committee members trained in the discipline. The content and views expressed in this thesis are those of the student-scholar and are not endorsed by Missouri State University, its Graduate College, or its employees. Follow this and additional works at: https://bearworks.missouristate.edu/theses Part of the Bioinformatics Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Abulibdeh, Amaal, "Investigation of the Homologs Rad51 and Dmc1 Role in Cell Division and Homologous Recombination" (2018). MSU Graduate Theses. 3269. https://bearworks.missouristate.edu/theses/3269 This article or document was made available through BearWorks, the institutional repository of Missouri State University. The work contained in it may be protected by copyright and require permission of the copyright holder for reuse or redistribution. For more information, please contact [email protected]. INVESTIGATION OF THE HOMOLOGS RAD51 AND DMC1 ROLE IN CELL DIVISION AND HOMOLOGOUS RECOMBINATION A Master's Thesis Presented to The Graduate College of Missouri State University TEMPLATE In Partial Fulfillment Of the Requirements for the Degree Master of Science, Cell and Molecular Biology By Amaal A. Abulibdeh May 2018 INVESTIGATION OF THE HOMOLOGS RAD51 AND DMC1 ROLE IN CELL DIVISION AND HOMOLOGOUS RECOMBINATION. Biomedical Sciences Missouri State University, May 2018 Master of Science Amaal A. Abulibdeh ABSTRACT RecA-like proteins homologs Rad51 and Dmc1 (disruption of meiotic control) promote recombination between homologous chromosomes by repairing programmed DNA Double-Strand Breaks (DSBs). Dmc1 is a Recombinase involved in meiosis-specific repair of DSBs, whereas Rad51 has been found to be involved in meiotic and non-meiotic DSBs repair. Previous studies showed that when RAD51 is overexpressed, interhomologous recombination still occurs even when DMC1 is knocked out. Dmc1 and Rad51 have not been fully characterized in the ciliate Tetrahymena thermophila. In order to more fully investigate the role of Rad51 and Dmc1 in Homologous Recombination Repair (HHR), this work focuses on using a model organism, T. thermophila, to further elucidate the contribution of Rad51 and Dmc1 in DNA repair following various genotoxic stressors (H2O2, MMS, and UV radiation). Bioinformatics was used to illustrate the extensive conservation of the Rad51 and Dmc1 homologs in various organisms and between one another. Expression of RAD51 and DMC1 was shown to be altered following exposure to H2O2, MMS, and UV radiation, and that the RAD51 expression was significantly higher than Dmc1 expression levels following all DNA damaging agents. Localization studies using Green fluorescent protein (GFP) and Red fluorescent protein (RFP) tagged to RAD51 or DMC1 and introduced back into T. thermophila revealed that Rad51 does not localize to the micronucleus or macronucleus following exposure to MMS. Tagging revealed that Dmc1 may localize in the micronucleus without DNA damage but does not localize after MMS treatment. Both proteins showed localization outside the nuclei, suggesting expression of the tagged Rad51 and Dmc1 in T. thermophila. KEYWORDS: Rad51, Dmc1, Homologous Recombination, DNA repair, Tetrahymena thermophila. This abstract is approved as to form and content _______________________________ Joshua J. Smith, PhD Chairperson, Advisory Committee Missouri State University ii INVESTIGATION OF THE HOMOLOGS RAD51 AND DMC1 ROLE IN CELL DIVISION AND HOMOLOGOUS RECOMBINATION. By Amaal A. Abulibdeh A Masters Thesis Submitted to the Graduate College Of Missouri State University In Partial Fulfillment of the Requirements For the Degree of Master of Sciences, Cell and Molecular Biology May 2018 Approved: _______________________________________ Joshua J. Smith, PhD _______________________________________ Colette M. Witkowski, PhD _______________________________________ Amanda C. Brodeur, MD, PhD _______________________________________ Julie Masterson, PhD: Dean, Graduate College In the interest of academic freedom and the principle of free speech, approval of this thesis indicates the format is acceptable and meets the academic criteria for the discipline as determined by the faculty that constitute the thesis committee. The content and views expressed in this thesis are those of the student- scholar and are not endorsed by Missouri State University, its Graduate College, or its employees. iii ACKNOWLEDGEMENTS First, I would like to express my sincere gratitude to my advisor Dr. Joshua J. Smith for the continuous support of my study, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my master study. Besides my advisor, I would like to thank the rest of my thesis committee: Dr. Amanda C. Brodeur, for her insightful comments and encouragement, and Dr. Colette M. Witkowski for steering me in the right direction whenever I needed it. I would like to thank the members of Smith Lab for their help during the research that went into this Thesis. I would especially like to thank Jeremy Tee for designing GFP and RFP epitope tags. Finally, I must express my very profound gratitude to my parents and to my husband, Amjed, for providing me with unfailing support and continuous encouragement throughout my years of study. This accomplishment would not have been possible without them. iv TABLE OF CONTENTS Introduction ..........................................................................................................................1 The Pathways of Double-Strand DNA Breaks ........................................................1 Non-Homologous End Joining ................................................................................3 Homologous Recombination Repair ........................................................................6 Eukaryotic Homologs of RecA: Rad51 and Dmc1 ..................................................9 Hop2-Mnd1 Complex ............................................................................................14 Tetrahymena thermophila as a Model Organism...................................................15 Meiosis in Tetrahymena thermophila ....................................................................17 The Recombinase Proteins and Hop2-Mnd2 Complex in Tetrahymena thermophila ......................................................................................19 Purpose Statement ..................................................................................................22 Materials and Methods .......................................................................................................23 Tetrahymena thermophila Strains and Growth Conditions ...................................23 Cryopreservation of Tetrahymena thermophila .....................................................23 LR Clonase™ Reaction .........................................................................................25 Electroporation Transformation of E.coli ..............................................................25 Midiprep DNA Purification ...................................................................................26 Biolistic Transformation of Tetrahymena thermophila .........................................27 Bioinformatics........................................................................................................29 qRT-PCR................................................................................................................30 Damage Treatment for RNA Extraction ................................................................32 Fluorescence Microscopy ......................................................................................33 Protein Isolation .....................................................................................................33 Results ...............................................................................................................................35 Bioinformatics........................................................................................................35 RAD51 and DMC1 Plasmid Purification and Transformation into Tetrahymena thermophila ......................................................................................38 Primer Optimization...............................................................................................49 RAD51 and DMC1 DNA Damage Expression after Various Damaging Agents 53 Fluorescent Microscopy of Rad51 and Dmc1 .......................................................54 Rad51 and Dmc1 Localization in Response to DNA Damage ..............................65 Discussion ..........................................................................................................................68 Bioinformatics........................................................................................................68 Expressions of RAD51 and DMC1 Following DNA Damage in Tetrahymena thermophila ............................................................................................................71
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