Investigating replication fork blocks, replication-transcription conflicts and replication restart dynamics in Escherichia coli

Abstract

DNA replication is essential for genome stability, but it is constantly jeopardized by various obstacles such as nucleoprotein complexes and transcription–replication conflicts. If not properly resolved, these impediments lead to replication fork collapse, genomic instability, and even cell death. This thesis investigates how Escherichia coli preserves its replication integrity using three experimental systems: site-specific protein– DNA blockades, engineered replication–transcription conflicts, and chemical stress induced by saccharin exposure. These studies were supported by the development of an automated bioimage informatics pipeline, utilizing deep-learning segmentation to enable high-throughput quantitative analysis of cellular morphology and SOS-induced stress phenotypes in live-cell time-lapse microscopy. Using a novel inducible fork-block model, I demonstrate that the PriA–PriB– DnaT pathway is the primary restart mechanism at nucleoprotein obstacles, with PriA helicase activity being essential for efficient replication restart. Surprisingly, when large tandem repeats were placed on the opposite replichore, PriC rather than PriB played the dominant role, raising the possibility that restart pathway usage is influenced by obstacle size or chromosomal context. Replication–transcription conflicts, generated by introducing an ectopic origin of replication (oriZ), similarly required PriA helicase and PriB for efficient fork restart. In their absence, cells displayed severe filamentation, heterogeneous stress phenotypes, and elevated Cas1–Cas2 foci. To further define the nature of these collisions, I utilized an alternative origin (oriX); the comparison between head-on and co-directional orientations confirmed that cellular pathology was specifically conflict- dependent. Genetic suppression with an RNA polymerase–destabilizing mutation confirmed that these defects stem directly from transcriptional collisions rather than indirect effects. Finally, I show that saccharin, a widely used artificial sweetener, induces replication stress in E. coli, with PriB-deficient cells exhibiting pronounced defects and loss of viability. These findings highlight how dietary compounds may disrupt gut microbial physiology. Collectively, this work establishes PriA helicase as a central player in replication restart and a promising antibacterial target. Since stalled fork rescue is also critical in cancer cells, these results also provide conceptual bridges between bacterial DNA replication and oncogene-induced replication stress, opening avenues for both antimicrobial and cancer therapeutic development.