Phenotypes of Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks.

The mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation The gene for the clamp loader subunits τ and γ, , but not the mutant , was found to suppress (Ts) when overexpressed. Purified mutant protein was found to be functional , and few phenotypes were found apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at never reach the terminus in mutant cells. The SOS response was found to be induced, and a combination of the mutation with and mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of and , respectively. The results indicate that the mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the -encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. The clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.