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Ribonuclease H Function in Bacillus Subtilis

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Ribonuclease H Function in Bacillus Subtilis Ribonuclease H Function in Bacillus subtilis by Justin R. Randall A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular and Developmental Biology) in The University of Michigan 2018 Doctoral Committee: Associate Professor Lyle A. Simmons, Chair Professor Ursula Jakob Assistant Professor Jayakrishnan Nandakumar Professor Nils Walter Justin R. Randall [email protected] ORCID iD: 0000-0002-5429-8995 © Justin R. Randall 2018 DEDICATION For my mother and father. Without your love and support this document, and come to think of it I myself, would not exist. ii ACKNOWLEDGEMENTS I’ve read that Oscar Wilde once said, “Success is a science; if you have the conditions, you get the result.” Oscar Wilde wasn’t a scientist, but in my humble opinion Lyle Simmons sets up pretty damn favorable conditions. From the moment I first met Lyle I knew I wanted to work in his lab. After introducing myself to him outside his office he said to me while I shook his hand, “I need to go to the bathroom want to come with me?” If I could go back I would have responded with a confident, “Sure.” But instead I awkwardly replied, “Umm…I think I’ll wait.” That was an awesomely odd first encounter which blossomed into a wonderful mentor/mentee and social relationship. If I were to try to engineer a dream Principle Investigator (PI), they would be to Lyle’s specs. I really could not have asked for a better mentor to lead me through my Ph.D. Thank you so much for all of your help and support throughout the last 5 years. I hope to one day develop your kindness, patience, humor, and scientific mind. To everyone else past and present who have called the Simmons lab home, I thank you for your shared knowledge, advice, and entertainment. Lenhart, I have never met someone who works harder than you. Brian, I leaned on your friendship and humor to get through rough science times. Jeremy, your mentorship and patience meant so much to my development as a scientist. Pete, iii I’ve never had a more confident and knowledgeable co-worker. Lindsay and Taylor, thank you for helping to keep me sane; whether through shared sushi and crossword solving or providing supportive advice when experiments weren’t going well. Katie and Lieke, though our time together was more brief, you both have very bright minds and I’m sure you will both be successful in the future. To Amy and Heather, thank you for putting up with my sarcasm and terrible humor. It was a pleasure having the opportunity to teach and spend time with you both. You are both terrific people and scientists. Good luck in your future endeavors! To my thesis committee, I can’t thank you enough for giving me your time and feedback. I know you are all very busy and I very much appreciate you making time to give me valuable feedback on my work and helping me stay on the correct path to complete my degree. I hope this document meets your expectations. To my friends, the last five and a half years would not have been nearly as fun nor gone by so fast without each of your friendships. Thank you all for the good times, too many drinks, disc golf, brewery tours, and general tomfoolery. I wish you all success in your new careers and hope to stay in touch throughout the years to come. To my family, I would have never made it this far without each of you. I became the person I am because of our shared experience, and I wouldn’t change a single thing about it. I love you all and hope I have made each of you proud. iv TABLE OF CONTENTS DEDICATION ......................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................... iii LIST OF FIGURES ...............................................................................................ix LIST OF TABLES .................................................................................................xi ABSTRACT...........................................................................................................xiii CHAPTER I. Introduction ...................................................................................................... 1 1.1 DNA/RNA structure ..................................................................................... 1 1.2 rNTP incorporation by DNA polymerases ................................................... 2 1.2.1 Replicative DNA polymerases ......................................................... 4 1.2.2 DNA polymerase I ............................................................................ 6 1.2.3 Y-family polymerases ...................................................................... 7 1.3 Primase and Okazaki fragments ................................................................. 8 1.3.1 Okazaki fragment maturation ........................................................... 8 1.4 R-loops ...................................................................................................... 10 1.5 RNase H family of enzymes ...................................................................... 11 1.5.1 RNase HI ....................................................................................... 12 1.5.2 RNase HII ...................................................................................... 13 1.5.3 RNase HIII ..................................................................................... 14 1.6 Ribonucleotide excision repair ................................................................... 15 v 1.6.1 Eukaryotic RER ............................................................................ 16 1.6.2 Bacterial RER ............................................................................... 17 1.7 Summary ................................................................................................... 18 1.8 Notes and Acknowledgements ................................................................... 18 II. Mutagenic cost of ribonucleotides in bacterial DNA ........................... 22 2.1 Abstract ...................................................................................................... 22 2.2 Significance ................................................................................................ 23 2.3 Introduction ................................................................................................. 24 2.4 Results ....................................................................................................... 27 2.4.1 RNase HII incises at single rNMPs in duplex DNA ....................... 27 2.4.2 RER-deficient B. subtilis cells accumulate GC → AT transitions .. 28 2.4.3 DNA polymerase I participates in RNase HII-dependent RER in B. subtilis ........................................................................................... 31 2.4.4 DnaE catalyzes error-prone resynthesis of a gapped substrate resulting in a G → A transition ...................................................... 32 2.4.5 Loss of NER alters the mutation spectrum in ΔrnhB ..................... 34 4.6 Discussion .................................................................................................. 35 4.6.1 Model for RER in B. subtilis. ......................................................... 35 2.6.2 Embedded ribonucleotides are unlikely to provide significant participation in nascent strand recognition during mismatch repair ...................................................................................................... 37 2.7 Materials and Methods ............................................................................... 38 2.7.1 RNase H cleavage assays ............................................................ 38 2.7.2 DNA polymerase activity assay .................................................... 39 2.7.3 Protein purification ........................................................................ 39 2.7.4 Calculation of conditional mutation rate ........................................ 41 2.7.5 Logistic regression of sequence context effect on transition rate .. 42 2.7.6 Motif identification at lagging strand template G to A transitions ... 42 vi 2.7.7 RNase HII nicked substrate extension .......................................... 43 2.7.8 Sequence context-dependent mutagenic resynthesis .................. 44 2.7.9 Gene reporter sequencing ............................................................ 45 2.7.10 Mutation accumulation line protocol .............................................. 46 2.7.11 Data-sharing plan ......................................................................... 47 2.7.12 Statistical analysis ........................................................................ 47 2.8 Notes and Acknowledgements .................................................................. 47 2.9 Figures and Tables .................................................................................... 48 III. Substrate specificity for bacterial RNase HII and HIII is influenced by metal availability .................................................................................... 62 3.1 Abstract ..................................................................................................... 62 3.2 Significance ............................................................................................... 63 3.3 Introduction ................................................................................................ 64 3.4 Results .....................................................................................................
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