UNDERSTANDING HUMAN DNA POLYMERASE EPSILON FLTNCTIONS: CANCER- ASSOCIATED MUTATOR VARIANTS, PROOFREADING DEFECTS AND POST. TRANSLATIONAL MODIFIC ATION S AN ABSTRACT SUBMITTED ON THE NINTH DAY OF MARCH 2015 TO THE DEPARTMENT OF BIOCI{EMISTRY AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF TI{E GRADUATE SCHOOL OF TULANE LINryERSITY FOR T}IE DEGREE OF DOCTOR OF PHILOSOPHY BY .\ -ru ./ -t' i..tr- -*<*fr**_-*f ERIN ELV ABETH T{ENNINGER APPROVED: F. Pursell, Ph.D. Advisor Dr. Samuel J. Dr. Victoria Bellancio ABSTRACT DNA Polymerase Epsilon (Pol ε) is one of three main eukaryotic Pols responsible for nuclear DNA replication. The Pol ε holoenzyme is comprised of four subunits, termed p261, p59, p17, and p12, with the largest subunit containing the DNA polymerase and 3ʹ to 5ʹ exonuclease (exo) proofreading activities. In addition to nuclear DNA replication, Pol ε participates in DNA repair, recombination, maintenance of epigenetic states and S-phase regulation, though the contribution of the smaller subunits to these processes is largely unknown. I set out to identify functions of the p12 subunit through determining post-translational modifications and protein- protein interaction partners. This approach found that p12 is likely constitutively phosphorylated and that p12 ubiquitylation dynamics may be important during replication stress and fork stalling. p12 also putatively interacts with proteins involved in maintaining genome stability including TOP1, HSP90, nucleolin and PRKDC. A larger portion of my project involved studying the role of cancer-associated mutations within the exo domain of POLE1, the gene encoding the p261 subunit. Tumors harboring these POLE1 mutations are hypermutated, with mutation frequencies exceeding 100 mutations/Mb. However, unlike POLE1 wild type hypermutated tumors, the POLE1 mutant tumors are microsatellite stable (MSS). With our collaborators at the Baylor College of Medicine Human Genome Sequencing Center and the Memorial Sloan Kettering Cancer Center, we determined that C→A and C→T base pair substitutions are highly elevated in these tumors relative to others, specifically at TCT and TCG motifs, respectively. I purified recombinant Pol ε and showed that several cancer mutant constructs, including S459F, P286H/R, L424V/I, and D275A/E277A, had elevated error rates for all 12 base pair substitutions and frameshifts, with the same propensity to make TCT→TAT mutations in vitro. In order to study the mechanism of how these specific mutations are made upon Pol ε exo inactivation in vivo, I constructed a knock-in cell culture model. In this model, I used targeted knock-in approach to introduce the D275A/E277A double amino acid substitution at the genomic POLE1 locus using an engineered recombinant adeno- associated virus. Mutation rates and base pair substitution error rates were both increased in a mismatch repair null background upon Pol exo inactivation. TCT→TAT basepair substitutions had the highest increase in error rate in the engineered cell culture system, as was seen in the POLE tumors, demonstrating the utility of this system for studying the relationship between Pol -dependent mutagenesis and tumor formation. The high rate of TCT→TAT mutagenesis has interesting consequences in tumors. The nucleotide preference of Pol ε variants leads to increases in recurrent nonsense mutations in key tumor suppressors such as TP53, ATM and PIK3R1. Moreover, strand-specific mutation patterns are seen during replication of these genes. Mapping of TCT→TAT hotspot mutations around known origin of replications provided the first direct evidence that Pol ε is the leading strand polymerase in human cells. The strand specificity of these mutations and high abundance in human tumors allows for unique identification of eukaryotic origins of replication. LINDERSTANDING HUMAN DNA POLYMERASE EPSILON FLINCTIONS: CANCER- ASSOCIATED MUTATOR VARIANTS, PROOFREADING DEFECTS AND POST- TRANSLATTONAL MODIFICATIONS A DISSERTATION SUBMITTED ON THE NTNTH DAY OF MARCH 2015 TO T}IE DE,PARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE GRADUATE SCHOOL OF TI.ILANE UNTVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY "' {*- .---*" l,..*4-**-:- ERIN ELIZABETH HENNINGER APPROVED: achfury F. Pursell, Dr. Victoria Bellanci<r ACKNOWLEDGEMENTS I first and foremost want to thank my advisor, Zachary Pursell, as without you this dissertation would not have been possible. I am thankful to have had the opportunity to study biochemistry in your lab, and I know you were very committed to giving me the best education I could get. To my committee members Dr. Victoria Belancio, Dr. Sam Landry, Dr. Arthur Lustig, and Dr. Astrid Engel: thank you all for being on my committee, and for the encouragements and acknowledgements you have given me during my stay at Tulane. I would also like to thank Dr. William Wimley and Dr. Erik Flemmington: you were great mentors to me. Thanks to my Pursell lab mates: Anderson Ayuk Agbor: my “big brother” who was very reassuring; Yassi Goksenin: the helpful raccoon who found fun new lab things for us to tinker with; Kim LeCompte: who gave great lab, baking, and shopping advice; Karl Hodel: who admirably works very hard and shares funny cat videos. To Dr. Paul Lambert and my labmates at Wisconsin: for helping me make the switch from chemistry to biology, being very patient with me and going out of their way to help me learn new things. To Wisconsin Friends: Soyeong Park and Rup Chakravorty: Thanks for all of the fun times we had together. I enjoyed our sleepover/study sessions in the Ebling Library and our weekly “BRIS” dinners at Nam’s Noodles. To Nicole Woodards, Shu Yao, and Justin Shorb: my chemistry buddies. Thank you for all the times we could be silly together and laugh until our sides hurt. We have too many fun adventures to name, and I hope to continue them. To Jessica Gross: It was such a joy spending undergrad and part of grad school with you. I’m so proud of what you’ve accomplished for your PhD; To Kimberly and George Dahlman: You are both ii amazing and strong people. Thanks for your support through a difficult time in my life. To Jim and Cheryl Rot: I love you guys so much (mom and dad!). I could write this whole section just on the two of you. And I don’t know where I would be without you. Thanks for helping me grow as a person, showing me what unconditional love is, and taking me in as your own daughter. Thanks to great friends from New Orleans, Philly, and Americorps: Brian Ridley: I admire your hard work and dedication to everything you do; Colleen Purcell: you have a wonderful, bubbly personality and you truly set an example with your passion for helping others; Kate Jenkins: thanks for getting me acquainted with New Orleans and being and awesome roommate and person; Andrea Covington: my twin and dancing buddy; Donna Edwards: thanks for all the laughs; Preet Gill: my best college friend, we had so many fun times together. I would even take P chem again if I could do it with you; Lesley McCall: I’m so lucky to have made a lifelong friend like you, and I have always looked up to you ever since I can remember; Cecilia Burns: I have never met a more kind and caring person, with such an endless store of compassion and I admire you so much; Rebecca Bortolin: you are one of my favorite people and a great concert buddy. I really appreciate all of your advice and compassion; Mallory Cortez: you are also one of my favorite people, an amazing scientist, and gave the best advice about grad school. I will miss our fun times at the camp and 80s nights; Teddy Livingston: you are so smart, considerate, and selfless. Thanks for helping me through difficult decisions and for being a great friend; Amie Devlin: my soulmate friend, I love that we have the best times when we are together, even if we were to end up stranded in NYC for a night, Your friendship has meant the world to me; Meredith Sosulski: I’m so glad we became such good friends. I will miss hanging out at Finns with you; Nam Nguyen: thanks for helping me through and always listening. You are a wonderful person who deserves great things; Brian Deskin: you were my first and are my best friend from BMS, and you are like a brother to me. Will see you soon in Europe; Aaron Miller: I know in heaven you are “in my corner.” I will always remember you. iii To the Nechvatal Clan, Grandma and Grandpa Henninger, and Uncle Brett: Thanks for always believing in me and loving me. I don’t know where I would be without any of you either, and it would take a long time to thank all of you, but thanks for teaching me how important family is. Uncle Brett, I know you supported me from heaven as well. I miss you. To Mom, Dad and Jeff: I know you would do anything for me, and I know I made it through because of you. I can’t thank you enough, for everything. I definitely have the best family one could ask for. I could go on and on, but I know I wouldn’t be able to thank you enough. I love you dearly. To my husband, Doug Stanley: I’m so glad I found you, and I know I would have even if I had stayed in Wisconsin. It was meant to be. I know it isn’t easy being with someone who has chronic illness, but it shows what an amazing and special person you must be. You truly are my best friend. Thank you beyond words.