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 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.