Replication of chromosomal DNA serves as the basis for genetic inheritance and occurs in all living organisms. However, the DNA replication machinery is known to face many cellular obstacles in the form of DNA-bound proteins or damage to the DNA template, which may impede the progression of the replication fork, or lead to its complete disassembly. This represents a serious threat to genome stability and cell viability if the stalled fork is not repaired, the replisome reloaded, and replication completed. In this work, we describe the use of a novel system for observing the inducible and reversible stalling of replication forks in live Escherichia coli cells. This fluorescent repressor operator system (FROS) allows for the assessment of replication status in real time, while also providing a mechanism for visualizing the structure of the DNA at the stalled or collapsed fork and the subsequent recombinational intermediates in the pathway towards replication restart.
A number of recombination proteins are known to act at stalled and collapsed replication forks, facilitating their repair and leading to restart. However, the specific roles, interactions and kinetics of the individual proteins remain poorly understood. Our approach uses E. coli strains deficient for a variety of key proteins and observes their capacity to process and restart replication following FROS-mediated stalling. We have shown that strains lacking single repair proteins, even the essential homologous recombination protein RecA, are able to restart replication rapidly following stalling. Other single knockout strains have exhibited an increase in the abundance of intermediate structures following stalling, suggesting a decreased ability to process stalled replication forks – highlighting novel roles of RecG and RecQ in replication fork reversal. Excitingly, these results suggest that the use of FROS to examine strains with multiple mutations will provide insight into the complex processes involved in rescuing stalled or collapsed replication forks.