The impact of the integrated stress response on DNA replication ____________________________________________________ Dissertation for the award of the degree "Doctor of Philosophy" (Ph.D.) Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen within the doctoral program Molecular Biology of Cells of the Georg-August University School of Science (GAUSS) submitted by Josephine Ann Mun Yee Choo from Selangor, Malaysia Göttingen 2019 Thesis Committee 1. Prof. Dr. Matthias Dobbelstein, Institute of Molecular Oncology, University Medical Center Göttingen (UMG) 2. PD Dr. Halyna Shcherbata, Research Group – Gene Expression and Signaling, Max Planck Institute for Biophysical Chemistry (MPI-BPC) 3. Prof. Dr. Steven Johnsen, Clinic for General, Visceral and Pediatric Surgery, University Medical Center Göttingen (UMG) Members of the Examination Board 1st reviewer: Prof. Dr. Matthias Dobbelstein, Institute of Molecular Oncology, University Medical Center Göttingen (UMG) 2nd reviewer: PD Dr. Halyna Shcherbata, Research Group – Gene Expression and Signalling, Max Planck Institute for Biophysical Chemistry (MPI-BPC) External members of the Examination Board 1. Dr. Roland Dosch, Department of Developmental Biochemistry, University Medical Center Göttingen (UMG) 2. Prof. Dr. Heidi Hahn, Department of Human Genetics, University Medical Center Göttingen (UMG) 3. Prof. Dr. Dieter Kube, Department of Hematology and Oncology, University Medical Center Göttingen (UMG) 4. Dr. Nuno Raimundo, Department of Cellular Biochemistry, University Medical Center Göttingen (UMG) Date of oral examination: 12th December 2019 This thesis is dedicated to my parents. TABLE OF CONTENTS Table of Contents 1 Abstract .......................................................................................................................................... 1 2 Introduction .................................................................................................................................... 2 2.1 Central dogma of Molecular Biology ........................................................................................... 2 2.2 Translational initiation – the rate limiting step of protein synthesis ............................................. 3 2.2.1 Cap-dependent initiation ..................................................................................................... 3 2.2.2 Cap-independent initiation ................................................................................................... 5 2.2.3 Signalling pathways affecting translation initiation .............................................................. 7 2.3 The integrated stress response .................................................................................................. 8 2.3.1 ISR sensors/regulators ........................................................................................................ 8 2.3.2 eIF2alpha phosphorylation inhibits cap-dependent protein synthesis ............................... 10 2.3.3 The ISR is active. What happens then? ............................................................................ 11 2.3.4 Termination of the ISR ...................................................................................................... 12 2.3.5 The ISR and cancer .......................................................................................................... 13 2.4 DNA replication......................................................................................................................... 13 2.4.1 Regulation of DNA replication ........................................................................................... 14 2.4.2 Replicative stress .............................................................................................................. 16 2.4.3 Replicative stress in cancer ............................................................................................... 16 2.5 Histones are essential for proper DNA replication .................................................................... 18 2.5.1 Regulation of replication-dependent histones ................................................................... 18 2.6 R-loops ..................................................................................................................................... 20 2.7 Scope of the study .................................................................................................................... 21 3 Manuscript ................................................................................................................................... 24 3.1 Abstract .................................................................................................................................... 25 3.2 Highlights .................................................................................................................................. 25 3.3 Significance .............................................................................................................................. 25 TABLE OF CONTENTS 3.4 Introduction ............................................................................................................................... 26 3.5 Methods and materials ............................................................................................................. 28 3.6 Results...................................................................................................................................... 36 3.7 Discussion ................................................................................................................................ 42 3.8 Acknowledgements .................................................................................................................. 45 3.9 Author contributions.................................................................................................................. 45 3.10 Declaration of Interests ............................................................................................................. 45 3.11 Figures/ Figure Legends ........................................................................................................... 47 3.12 Supplementary Figures/ Supplementary Figure Legends ........................................................ 63 4 Discussion .................................................................................................................................... 82 4.1 Interplay between the ISR and DNA replication ....................................................................... 83 4.1.1 DNA replication is inhibited upon ISR stimulation ............................................................. 83 4.1.2 The ISR does not activate replicative stress signalling ..................................................... 84 4.2 Crosstalk between DNA replication and protein translation ..................................................... 85 4.2.1 The processes of DNA and protein synthesis are co-regulated ........................................ 85 4.2.2 DNA replication proteins have long half-lives .................................................................... 86 4.3 Histones: a limiting factor in DNA replication ............................................................................ 87 4.3.1 Continuous histone synthesis ensures proper DNA replication ........................................ 87 4.3.2 ISR depletes cells of histones ........................................................................................... 87 4.3.3 Histone overexpression restores DNA replication upon ISR ............................................. 89 4.4 R-loops accumulate with the ISR ............................................................................................. 90 4.4.1 The ISR blocks DNA replication through R-loops independent of ATF4 ........................... 90 4.4.2 R-loops are enriched upon histone depletion .................................................................... 92 4.4.3 R-loops formed upon ISR are not threats to genomic stability .......................................... 94 4.5 Therapeutic potential of ISR in cancer ..................................................................................... 95 4.5.1 Activating the ISR to block DNA replication and proliferation in cancer ............................ 95 4.5.2 Inhibiting the ISR to suppress tumourigenesis .................................................................. 97 TABLE OF CONTENTS 4.5.3 Modulating the ISR in combination with other therapies ................................................... 99 4.6 Conclusions and future perspectives ...................................................................................... 100 5 References ................................................................................................................................. 102 6 Appendix .................................................................................................................................... 117 7 Abbreviations ............................................................................................................................. 121 8 List of Figures ............................................................................................................................ 127 9 Acknowledgements .................................................................................................................... 129 10 Curriculum