DISSERTATION THE CHROMATIN BINDING FACTOR SPN1 CONTRIBUTES TO GENOME INSTABILITY IN SACCHAROMYCES CEREVISIAE Submitted by Alison K. Thurston Department of Biochemistry and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2018 Doctoral Committee: Advisor: Laurie Stargell Susan Bailey Jennifer DeLuca Jeffrey Hansen Karolin Luger Copyright by Alison K. Thurston 2018 All Rights Reserved ABSTRACT THE CHROMATIN BINDING FACTOR SPN1 CONTRIBUTES TO GENOME INSTABILITY IN SACCHAROMYCES CEREVISIAE Maintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a “healthy level” of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently ii promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle. iii ACKNOWLEDGEMENTS Obtaining a PhD has been a long hard journey, and while the research is independent, I did accomplish it alone. First, I would like to thank my advisor, Dr. Laurie Stargell. I respect her outlook on research, education and our responsibility to the public. Not only has she guided my intellectual approach to research but she stresses the importance of communication to fellow scientists and the general public. In the future, I will approach my writing and public speaking as if she is there guiding me and telling me to keep it simple. Her generosity and encouragement allowed me to pursue opportunities which were important for success in graduate school and for my future career. To my laboratory members: Dr. Cathy Radebaugh, Dr. Xu Chen, and Dr. Lillian Huang; thank you for sharing your wisdom with me. I have enjoyed working with you every day and hope that in the future I will always be lucky enough to work in such a caring, motivating, talented and inspiring environment. I would like to thank my graduate committee: Dr. Susan Bailey, Dr. Jennifer DeLuca, Dr. Karolin Luger and Dr. Jeffrey Hansen. I appreciate the time and guidance you have all contributed to my success. Jeff, I would like to thank you specifically for your encouragement and perspective. I truly valued working with you and appreciate the opportunities to teach in your classes. Additionally, I would like thank the Biochemistry and Molecular Biology Department and Colorado State University. Furthermore, I would like to acknowledge Dr. Juan Lucas Argueso. I am grateful for the time and conversations spent answering my questions, advising me on experimentation, and your insight into my project. iv I would like to thank my fellow Stargell Laboratory graduate students: Dr, Sha Li and Dustin Steele. I will miss our conversations and the comradery and I am excited for all of our futures. Furthermore, I cannot image graduate school without Jen Shattuck, Dr. Melissa Ford and Sarah Bollinger. As colleagues, classmates and friends, you all made the journey much more enjoyable. I am grateful to Dr. Robyn Barbato, the Barbato Laboratory members, the Cold Regions Research and Engineering Laboratory (CRREL) and the DHS-STEM program for the opportunity, welcoming environment and funding for my research internship. Robyn, you are great mentor and I appreciate the freedom granted to pursue research in your laboratory. To my family, I am thankful for all your support and belief in me. The visits and care packages were needed pick me ups. I know that you will always be there. Perhaps now I will have time for a massage. Finally, I would like to thank Eric. You were there night and day. I am glad and thankful that you decided to take this journey with me. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................. ii ACKNOWLEDGEMENTS .......................................................................................................... iv CHAPTER 1: REVIEW OF THE LITERATURE ......................................................................... 1 1.1 Genome Stability/Instability .............................................................................................. 1 1.2 DNA Damage Repair Pathways ........................................................................................ 1 1.3 Assessment of Genome Instability .................................................................................... 3 1.4 Chromatin and Genome Instability .................................................................................... 6 1.5 SPN1 ................................................................................................................................ 8 CHAPTER 2. MATERIALS AND METHODS ............................................................................11 2.1 Yeast Strains and Culturing .............................................................................................11 2.1.1 Culturing ...................................................................................................................11 2.1.2 Spn1 mutants in deletion strains ...............................................................................11 2.1.3 Phosphorylation mutants ...........................................................................................11 2.1.4 Loss of heterozygosity ..............................................................................................12 2.2 Phenotypic Assays ..........................................................................................................12 2.3 Fluctuation Analysis .........................................................................................................12 2.4 Loss of Heterozygosity Assay ..........................................................................................13 2.5 Budding Index .................................................................................................................13 2.6 Immunoblotting Analysis ..................................................................................................14 2.7 Micrococcal Nuclease Digestion ......................................................................................14 2.8 Indirect End Labeling .......................................................................................................15 2.9 Spn1 Molecules per Cell ..................................................................................................15 2.10 Flow Cytometry ..............................................................................................................15 2.11 Chronological Aging Assay ............................................................................................16 CHAPTER 3. SPN1 CONTRIBUTES TO GENOME INSTABILITY ...........................................24 3.1 Summary .........................................................................................................................24 3.2 Introduction ......................................................................................................................24 3.3 Results ............................................................................................................................27 3.3.1 Expression of spn1141-305 results in cellular resistance to methyl methanesulfonate ...27 3.3.2 Removal of methyl lesions through Mag1 glycosylase is necessary for resistance. ...28 3.3.3 Resistance to MMS is independent of the nucleotide excision repair pathway ..........31 vi 3.3.4 Resistance is dependent on the error free sub-pathway of the DNA damage tolerance pathway .............................................................................................................................31 3.3.5 Spn1 contributes to spontaneous and damage induced genome instability. ..............33 3.3.6 Resistance