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Restricted by Xercd Sister-Chromosome Resolvase RICE UNIVERSITY Bacterial Fragile Sites and Mutational Patterns of Human ‘Damageome’ Proteins By Mei, Qian A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Doctor of Philosophy APPROVED, THESIS COMMITTEE Jonathan Silberg Jonathan Silberg (Aug 7, 2020 09:21 CDT) Susan Rosenberg (Aug 12, 2020 13:01 CDT) Jonathan Silberg (Chair) Susan M. Rosenberg (Director) Professor Professor Biosciences, Rice University Molecular and Human Genetics, Baylor College of Medicine Yousif Shamoo Professor Biosciences, Rice University Jane Grande-Allen Professor Bioengineering, Rice University HOUSTON, TEXAS August 2020 Abstract Genome instability fuels cancer, genetic diseases and evolution. Fragile sites are a source of genome instability, and are defined as regions of recurrent DNA damage in genomes. Although associated with many human diseases, the basis of fragility is still not fully understood. Holliday junctions (HJs) are four-way DNA junctions that form as intermediates in DNA repair and as a type of DNA damage when stalled replication forks collapse, and therefore are markers of fragility. Here, I identify three bacterial fragile sites of recurrent HJs located in the replication terminus (Ter) region of the Escherichia coli genome. I show that formation of HJs require DNA double-strand break (DSB) repair factors and are associated with recurrent DSBs, detected by DNA-end sequencing (END-seq). Two mechanisms that contribute to the three fragile sites are revealed. Due to the conserved nature of DNA transactions, my findings may help explain important disease-relevant unstable sites in human. In a separate study, the recently discovered DNA “damageome” proteins (DDPs) promote endogenous DNA damage when overproduced, and thus constitute another source of genome instability. Many DDPs and at least three DDP mechanisms have been found in E. coli using high-throughput methods. Despite the strong association of human DDP homologs with cancer-mutations, the study with experimental methods is more challenging. Here in this thesis, I mine public cancer databases to explore potential DDP roles in promoting mutations that drive cancers, to guide the experiments. Finally, the cancer chemotherapy drug 5-fluorouracil (5-FU) works by destabilizing ii the genome through the induction of thymineless death (TLD). We leverage the knowledge of E. coli TLD resistance genes to discover novel 5-FU-resistance genes in cancers. iii Acknowledgments I would like to thank many great scientists who inspire me to go to graduate school and pursue a career as a scientist. Many thanks to my mentor, a distinguished scientist, and a good friend, Susan Rosenberg. I thank Susan for admitting me to the lab when I was a ‘dry lab’ student and had almost no experience in experimental biology. She motivates me to dream big, go beyond the scope to take on challenging project, and she provides full support when I encounter obstacles and setbacks. This work could never have been accomplished without her. I am grateful to P.J. Hastings for his help on my writing, and pushing me to dive deep into science. It is always a pleasure to discuss science and life with him, and listen to his stories. I have been fortunate enough to spend years with a group of smart and great people in the Rosenberg and Hastings lab. They are very kind and helpful. I thank them, and really appreciate the supportive environment created by them. I thank my graduate program, my advisory and thesis committee, and the administrative team for ensuring that I have a pleasant graduate school experience. Last, and most importantly, I thank my family, especially my parents, for their unconditional support. They respect my decisions, lift my spirit, and encourage me to follow my heart. iv Contents Abstract ...................................................................................................................... ii Acknowledgments ...................................................................................................... iv Contents ...................................................................................................................... v List of Figures ............................................................................................................. vii List of Tables ............................................................................................................... x Nomenclature ........................................................................................................... 11 Introduction .............................................................................................................. 12 DNA-junction Landscape Reveals Chromosome-fragility Mechanisms in Bacteria ...... 20 2.1. Summary ............................................................................................................. 21 2.2. Introduction .......................................................................................................... 22 2.3. Results ................................................................................................................. 22 2.3.1. Holliday junction map reveals fragile sites ............................................... 22 2.3.2. Recurrent DSBs near terminal HJs ........................................................... 25 2.3.3. Replication-fork barriers as fragile sites ................................................... 26 2.3.4. Chromosome segregation failure and fragility ......................................... 28 2.3.5. Fragility is common and fragile sites interact .......................................... 31 2.4. Implications ......................................................................................................... 32 2.5. Supplemental Discussion .................................................................................. 34 2.6. Methods ............................................................................................................... 36 2.6.1. Strains, media and growth ......................................................................... 36 2.6.2. X-seq and ChIP-seq library preparation and sequencing ...................... 36 2.6.3. END-seq library preparation and sequencing ......................................... 38 2.6.4. Analysis of sequencing data ...................................................................... 40 2.6.5. Detecting peak boundaries in X-seq data ................................................ 41 2.6.6. Statistics ........................................................................................................ 41 2.6.7. Data availability ............................................................................................ 41 2.7. Figures ................................................................................................................. 42 v 2.8. Extended Data Figures ...................................................................................... 50 2.9. Supplementary Table ......................................................................................... 65 2.10. References ........................................................................................................ 68 Mutation Analysis Implicates the Roles of Candidate Human DNA “Damagome” Proteins in Cancers .................................................................................................... 73 3.1. Summary ............................................................................................................. 73 3.2. Introduction .......................................................................................................... 74 3.3. Results ................................................................................................................. 75 3.4. Discussion ........................................................................................................... 80 3.5. Methods ............................................................................................................... 81 3.6. Figures and Tables ............................................................................................ 82 3.7. References .......................................................................................................... 99 Deep Translational Discovery of Cancer Chemotherapy Resistance Genes ............... 101 4.1. Summary ........................................................................................................... 101 4.2. Introduction ........................................................................................................ 103 4.3. Results ............................................................................................................... 104 4.4. Conclusions ....................................................................................................... 105 4.5. Methods ............................................................................................................. 106 4.5.1. Targeted next-generation sequencing analysis .................................... 106 4.5.2. TCGA primary tumor files analysis ......................................................... 107 4.6. Tables ................................................................................................................ 108 4.7. References ........................................................................................................ 136 Conclusions and Future Directions ..........................................................................
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