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The Next Generation the Development and Application of Improved Methods for Assessment of Replisome Protein Dynamics University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Ipond2: The Next Generation The Development And Application Of Improved Methods For Assessment Of Replisome Protein Dynamics Rebecca Rivard University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Rivard, Rebecca, "Ipond2: The Next Generation The Development And Application Of Improved Methods For Assessment Of Replisome Protein Dynamics" (2017). Publicly Accessible Penn Dissertations. 2552. https://repository.upenn.edu/edissertations/2552 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2552 For more information, please contact [email protected]. Ipond2: The Next Generation The Development And Application Of Improved Methods For Assessment Of Replisome Protein Dynamics Abstract Faithful replication of the genome during cell division is essential for the avoidance of disease-promoting mutations. Until recently, accurate quantification of eplicationr factor alterations in response to cellular stress primarily relied on low sensitivity assays such as cell staining and nuclear extraction assays. In the past few years, the Cortez, Groth, and Santocanale laboratories have developed procedures to retrieve and analyze proteins associated with actively replicating DNA (iPOND, NCC, and Dm-Chp, respectively). Herein, we report improvements to iPOND that increase protein yield and quantitative sensitivity, as well as permit better statistical evaluation of candidate factors (iPOND2). These improvements were achieved by employment of sucrose based density gradient fractionation of samples prior to EdU-biotin retrievals. The use of iPOND2 increased the dynamic range of protein quantification yb Mass Spec by more than 40-fold compared to recent iPOND. We investigated the replisome component response to stress and assessed the role of p97-mediated degradation in protein turnover at the fork with or without cell cycle checkpoint protein, ATR. Furthermore, increased replisome component yields permitted the detection of ubiquitylated peptides without secondary affinity-based retrievals. Due to the increased yield of iPOND2 we were able to combine iPOND2 with other purification methods such as -K ɛ-GG IP to gain further utility from the addition of sucrose fractionation to iPOND. For example, we have further improved our ability to analyze ubiquitin sites on the replisome in a high-throughput way and potentially developed a method capable of assessing terminated fork or origin composition across a variety of treatment conditions. In summary, iPOND2 exhibits greatly improved replisome retrieval specificity, yield, quantitative dynamic range, and statistical power to detect changes in replication-associated factors in response to stress conditions. iPOND2 can be used alone, in conjunction with a variety of MS analytical methods, and combined with secondary affinity purificationso t improve our understanding of replisome dynamics following stress. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Eric Brown Second Advisor Brad Johnson Keywords ATR, iPOND, Replication, Replisome Subject Categories Cell Biology | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2552 iPOND2: THE NEXT GENERATION THE DEVELOPMENT AND APPLICATION OF IMPROVED METHODS FOR ASSESSMENT OF REPLISOME PROTEIN DYNAMICS Rebecca S. Rivard A DISSERTATION In Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation _______________________ Eric J. Brown, Ph.D. Associate Professor of Cancer Biology Graduate Group Chairperson ________________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Brad Johnson, MD, Ph.D., Associate Professor of Pathology and Laboratory Medicine Luca Busino, Ph.D., Assistant Professor of Cancer Biology Benjamin Garcia, Ph.D., Presidential Professor of Biochemistry and Biophysics and Epigenetics Paul Lieberman, Ph.D., Wistar Institute Professor of Microbiology DEDICATION PAGE This thesis is dedicated to the souls of all the graduate students that were lost in the pursuit of knowledge. And to my family and husband who were essential in saving me from becoming one of them. To them I will be eternally grateful. ii ACKNOWLEDGEMENTS Kasia Kulej, Anja Bielinsky, Ya-chu Change, and Yee-mon Thu: Thank you for teaching me about MS and bioinformatics. Brad Johnson, Ben Garcia, Luca Busino, and Paul Lieberman: Thank you for all of your support and feedback. Eric Brown, Ryan Ragland, Sara Small, Sima Patel, Jessica Tang, David Shang, Laura Murillo, Yu-Chen Tsai, Nishita Shastri: Thank you for your input on my projects, help with the eternal edits and revisions of my paper, and support. Lydia Mitchleson, Amanda Limas, Miles Abidila, Aseem Mulji: Thank you so much for helping to cheer me up when I needed it. Lizzie Bouton, Jaci Rifkin, and Mymy Simckes: You are some of my oldest friends who have helped me gained a lot of the strength required to complete this program and helped motivate me when I did not feel that I could to it on my own. Moose: Thank you for all of the cuddles and some much needed judgement. Robin Rivard, Mike Rivard, Bill Miles, Pat Smiley, Emma Rivard, and Abby Rivard: Thank you for being a wonderful and supportive family all the way through this process, and always lending a helping hand when I needed one. Theonie Anastassiadis: Thank you so much for being my bay mate and work wife. I could not have made it through the last few years without you as a constant source of help, smiles, support, and advice. I loved getting to travel the world with you and am so grateful that I have had you with me throughout this experience. You gave me a reason to come into work and I will miss getting to see you every day. No matter where we go in the future the Ebola Babes will always live on in our hearts. Tim Miles: Thank you for being the best husband ever. Thank you for always knowing what to say to cheer me up and loving and supporting me unconditionally. No matter how bad things got you were always there to help pick me back up again. Thank you for forcing me to take breaks when I need them and taking care of me. I love you so much and cannot thank you enough for everything that you have done for me, nor express in words what you truly mean to me. Thank you for being my everything. iii ABSTRACT iPOND2: THE NEXT GENERATION THE DEVELOPMENT AND APPLICATION OF IMPROVED METHODS FOR ASSESSMENT OF REPLISOME PROTEIN DYNAMICS Rebecca S. Rivard Eric J. Brown Faithful replication of the genome during cell division is essential for the avoidance of disease-promoting mutations. Until recently, accurate quantification of replication factor alterations in response to cellular stress primarily relied on low sensitivity assays such as cell staining and nuclear extraction assays. In the past few years, the Cortez, Groth, and Santocanale laboratories have developed procedures to retrieve and analyze proteins associated with actively replicating DNA (iPOND, NCC, and Dm-Chp, respectively). Herein, we report improvements to iPOND that increase protein yield and quantitative sensitivity, as well as permit better statistical evaluation of candidate factors (iPOND2). These improvements were achieved by employment of sucrose based density gradient fractionation of samples prior to EdU-biotin retrievals. The use of iPOND2 increased the dynamic range of protein quantification by Mass Spec by more than 40-fold compared to recent iPOND. We investigated the replisome component response to stress and assessed the role of p97-mediated degradation in protein turnover at the fork with or without cell cycle checkpoint protein, ATR. Furthermore, increased replisome component yields permitted the detection of ubiquitylated peptides without secondary affinity-based retrievals. Due to the increased yield of iPOND2 we were able to combine iPOND2 with other purification methods such as K-ɛ-GG IP to gain further utility from the addition of sucrose fractionation to iPOND. For example, we have further improved our ability to analyze ubiquitin sites on the replisome in a high-throughput way and potentially iv developed a method capable of assessing terminated fork or origin composition across a variety of treatment conditions. In summary, iPOND2 exhibits greatly improved replisome retrieval specificity, yield, quantitative dynamic range, and statistical power to detect changes in replication-associated factors in response to stress conditions. iPOND2 can be used alone, in conjunction with a variety of MS analytical methods, and combined with secondary affinity purifications to improve our understanding of replisome dynamics following stress. v Table of Contents DEDICATION PAGE ................................................................................................................ II ACKNOWLEDGEMENTS ..................................................................................................... III ABSTRACT ............................................................................................................................... IV LIST OF ILLUSTRATIONS ..................................................................................................
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