DNA Damage and Base Excision Repair; An Important Role during Zebrafish Embryogenesis. By Stephen Paul Gifford Moore B.S. in Biology, University of Auckland P.G.Dip.Sci, in Marine Science, University of Auckland M.S. in Marine Science, University of Auckland A dissertation submitted to The Faculty of the College of Science of Northeastern University in partial fulfilment of the requirements for the degree of Doctor of Philosophy December 1st, 2017 Dissertation directed by Phyllis R. Strauss 1 Dedication Dedicated to the memory of my father, Michael Douglas Gifford Moore. December 1944 to March 2013. Gone, but not forgotten. 2 Acknowledgements This dissertation represents over five years of extremely hard work, some of which I did. First and foremost, I wish to extend my deepest gratitude to my advisor, Dr Phyllis Strauss, for giving me the opportunity to join her lab. I learnt so much during my dissertation, and have become a more educated, confident, and responsible scientist and person. I owe this to Dr. Strauss’ unwavering patience, support, and mentorship over the years. I would also like to thank Dr Erin Cram, Dr Veronica Godoy, and Dr James Monaghan, from the Northeastern University Department of Biology, who were members of my dissertation committee. I am especially grateful for all the help and advice I received on my research goals, and help keeping me focused on end goals. I would especially like to thank the fifth member of my committee, Dr Leona Samson of the Center for Environmental Health Sciences at MIT, for her mentorship and advice over the years. I would like to save a very big and special thank you to all the past members of the Strauss lab for their companionship and help. I would especially like to thank Ben Snow for the research that he did as part of his M.S. His findings were instrumental in guiding the direction that I took my own research in. Also, thank you to Kim Toomire and Josh Kruchten, who both did research projects on my behalf that we managed to publish. Thanks to Brian Dobosh too, who also did an amazing job with some qPCR work, that unfortunately did not end up being used. I also want to say thank you to everyone in the Cram lab, where I spent a great deal of time in my final year and half at Northeastern. All your humor, friendship and helpful advice inspired me to continue in my work, despite frustrations. Thank you to the Biology Department of Northeastern University, particularly Dr Wendy Smith for all the work in the background that she did and for providing me with bench space to work at once our lab had closed. Thanks to Adrian Gilbert who made stuff happen, and Frauke Argyros who made teaching Biochemistry labs much more tolerable. A big thank you to all my fellow graduate students in the Biology department over the years who made my time at Northeastern University all that more enjoyable. 3 And finally, thank you to all my friends and family who supported me unwaveringly during a very busy and difficult time. A very special chur bro to my wife Maria who had to make so many sacrifices over the years while I chased rainbows and hunted unicorns, thank you! The final thank you goes to my daughter Ada, thank you for being you. 4 Abstract of Dissertation Damage to DNA is an unavoidable consequence of life. Unrepaired DNA damage is mutagenic, promotes genomic instability, and leads to the onset of numerous serious diseases. Multiple pathways have evolved to repair specific types of DNA damage. Of these, the multi- enzyme base excision repair (BER) pathway is considered the most active, because BER repairable damage is ongoing. Embryogenesis is a highly complex and regulated process. Since developing embryonic cells divide rapidly, unrepaired DNA damage leads to mutation and cell death. Many BER enzymes are embryonic lethal as knockouts, but not in adult cell cultures. This indicates an important role during embryonic development. Active DNA demethylation of 5-methylcytosine (5mC) in CpG islands during embryogenesis may result in DNA damage requiring BER. AP endonuclease (Apex1) is an essential BER enzyme. It incises abasic (AP) sites resulting from removal of DNA lesions by DNA glycosylases, allowing subsequent repair. Accumulation of AP sites is toxic. Knockdown (K/D) of Apex1 in zebrafish embryos results in a consistent and concurrent loss of the critical transcription factor (TF) Creb1. Many Creb1 dependent genes are subsequently perturbed resulting in abnormal embryo development. Death occurs at ~7 days post fertilization (7dpf). Research in this dissertation shows that DNA damage increases in zebrafish embryos at the mid-blastula transition (MBT) when zygotic genome activation (ZGA) occurs. Damage is further elevated with Apex1 K/D, thereby illustrating the importance of fully functional BER during embryonic development. Furthermore, increased DNA damage inversely correlates with decreased levels of 5mC and vice versa, providing indirect evidence that active DNA demethylation is at least one source of elevated embryonic DNA damage. The Creb1 binding site (CRE site [TGACGTCA]) is present within the promoter CpG island of the creb1 gene and some 5000 other genes. It is likely that active DNA demethylation of the Creb1 promoter may also affect the CRE site CpG dinucleotide. My published work shows that binding of recombinant CREB1 to the CRE site is modulated by DNA damage, and abolished by 5mC in the CpG. We also find that both recombinant CREB1 and BER glycosylases compete when DNA damage occurs within a TFs binding sequence, and demonstrably affects embryo development. These novel findings provide insight into the presence, timing, and potential source of DNA damage during zebrafish embryogenesis. They also show how DNA damage may act in an epigenetic fashion when it occurs in a TF binding site, and that these results are valid both in vitro and in vivo. We speculated that BER substrates act in an epigenetic fashion. 5 Table of Contents Dedication ................................................................................................................................. 2 Acknowledgements .................................................................................................................. 3 Abstract of Dissertation ........................................................................................................... 5 Table of Contents ..................................................................................................................... 6 List of Figures ........................................................................................................................... 8 List of Tables ............................................................................................................................ 9 Chapter 1. Introduction ........................................................................................................ 10 1.1. DNA damage and repair systems ........................................................................................... 10 1.1.1. Nucleotide excision repair .................................................................................................. 11 1.1.2. Mismatch repair .................................................................................................................. 12 1.1.3. Homologous recombination (HR) and non-homologous end joining (NHEJ) ................... 12 1.1.4. Base excision repair ............................................................................................................ 13 1.2. AP endonuclease....................................................................................................................... 14 1.2.1. Non-endonuclease function of Apex1 ................................................................................ 16 1.3. Zebrafish as a model system ................................................................................................... 17 1.4. Base excision repair and embryonic development ................................................................ 17 1.4.1. Apex1 studies and zebrafish embryogenesis ...................................................................... 18 1.5. DNA methylation ..................................................................................................................... 19 1.5.1. DNA methylation ................................................................................................................ 20 1.5.2. DNA demethylation ............................................................................................................ 20 1.5.3. DNA methylation and embryogenesis ................................................................................ 21 Chapter 2. DNA damage and cytosine methylation status are temporally modulated during zebrafish embryogenesis. .......................................................................................... 26 2.1. Introduction .............................................................................................................................. 27 2.2. Experimental procedures ........................................................................................................ 29 2.3. Results ....................................................................................................................................... 32 2.4. Discussion ................................................................................................................................. 35 Chapter 3. DNA