DNA replication termination and chromosome dynamics in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪

Abstract

Faithful genome duplication requires accurate replication initiation, elongation, and termination. In Escherichia coli, replication initiates bidirectionally from oriC and concludes when converging forks meet in the termination region. Recent work from the Rudolph lab has shown that this fusion process is risky and can jeopardize stability of the termination area. E. coli’ s chromosome contains a specialized “replication fork trap” comprised of Tus binding ter sites to direct termination events into a con􀏐ined region. However, the function and evolution of this fork trap system is unclear, as are the key proteins and pathways needed to complete replication. This work investigates chromosome architecture and impacts of fork fusions on genomic instability in E. coli by measuring recombination at de􀏐ined chromosome locations and accumulation of R‑loops that threaten genomic stability. Bioinformatics revealed maintenance of the fork trap structure across diverse E. coli strains and related enterobacteria, and that inactivation of Tus leads to mild growth defects, showing that while not all bacteria have a fork trap system, it is maintained once acquired leading to advantageous growth. Ectopic replication fusion events increased local recombination rates, implying fork collisions threaten genomic stability, with RecG and UvrD mutants showing synergistic effects of DNA metabolism in the termination area. R‑loop analysis demonstrated Tus synergizes with accessory helicases RecG and UvrD to limit global R‑loop accumulation, and I suggest the R‑loop toxicity begins in the termination area. Unrestrained R‑loop buildup induced toxicity over time, highlighting the danger of unrestricted R‑loop accumulation and importance of enzymatic processing by RecG and UvrD. These 􀏐indings provide key insights into replication termination in E. coli. They suggest the fork trap and repair pathways helps contain deleterious consequences of fork fusions, while promoting proper chromosome segregation. This work establishes principles relevant to both prokaryotes and eukaryotes regarding maintenance of genomic integrity during the essential process of DNA replication. Elucidating bacterial systems that enable faithful completion of replication enhances fundamental understanding of chromosome duplication mechanisms required for genome stability across all domains of life.